UNIVERSITY of CALIFORNIA, SAN DIEGO the Intricacies of UGT

UNIVERSITY OF CALIFORNIA, SAN DIEGO

The Intricacies of UGT Regulation:

Protein-Protein Interactions and Environmental Arsenic Exposure

A dissertation submitted in partial satisfaction of the requirements for the degree of

Doctor of Philosophy

Chemistry

Camille Maria Konopnicki

Committee in charge:

Professor Robert H. Tukey, Chair Professor Pieter Dorrestein Professor James Halpert Professor Alexander Hoffmann Professor William Trogler

2012

Camille Maria Konopnicki, 2012

The Dissertation of Camille Maria Konopnicki is approved, and it is acceptable in quality and form for publication on microfilm and electronically:

______

______Chair

University of California, San Diego

2012

iii DEDICATION

To my parents, Marek Konopnicki and Barbara Pawlowski-Konopnicki

and my family, past and present.

iv EPIGRAPH

Play is the highest form of research.

Albert Einstein

v TABLE OF CONTENTS

SIGNATURE PAGE ...... iii

DEDICATION ...... iv

EPIGRAPH ...... v

TABLE OF CONTENTS ...... vi

LIST OF ABBREVIATIONS ...... viii

LIST OF FIGURES...... xii

LIST OF TABLES ...... xiv

ACKNOWLEDGEMENTS ...... xv

VITA ...... xx

ABSTRACT OF THE DISSERTATION...... xxiii

CHAPTER 1 Introduction to Drug Metabolism ...... 1 Drug Metabolizing Enzymes from an Evolutionary Perspective...... 2 The Function of Phase I and Phase II Drug Metabolizing Enzymes...... 3 Characterization of the Glucuronidation Process...... 4 UDP-Glucuronsyltransferases ...... 6 Tissue-Specific Expression of the UGTs ...... 12 Individual UGT Variability, Disease, and Adverse Drug Reactions...... 14 Protein-Protein Interactions Between DMEs ...... 20 UGT-UGT Interactions ...... 22 RNA Interference as a Tool to Study Glucuronidation...... 24 The Xenobiotic Receptors ...... 27 The Aryl Hydrocarbon Receptor...... 29 Nuclear Receptors ...... 33 The Pregnane X Receptor and the Constitutive Androstane Receptor...... 37 The Oxidative Stress Sensor, NF-E2 related factor-2 ...... 42 The UGT1A1 Phenobarbital Response Enhancer Module ...... 49 The NF-κB/IKK Pathway...... 50 The Mitogen Activated Protein Kinases...... 55

vi Objectives of the Dissertation ...... 61 References ...... 65

CHAPTER 2 Evaluation of UGT Protein Interactions in Human Hepatocytes: Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on Propofol Glucuronidation in Human Hepatocytes ...... 112 Introduction ...... 113 Experimental...... 115 Results ...... 120 Propofol Glucuronidation in Recombinant UGTs ...... 120 siRNA Characterization ...... 121 Inhibition of Propofol Glucuronidation by siRNA Down Regulation in Human Hepatocytes ...... 127 Discussion...... 128 References ...... 132

CHAPTER 3 The Regulatory Role of Oral Arsenic in hUGT1 Mice ... 137 Introduction ...... 138 Humanized Mice as a Sensor for Environmental Toxicant Exposure ...... 138 Environmental Arsenic Contamination...... 140 Arsenic-Induced Generation of Reactive Oxygen Species ...... 143 Arsenic Can Modulate Xenobiotic Transcriptional Activation...... 146 Arsenic Impacts the NF-κB/IKK Signaling Pathway ...... 149 Arsenic Influences the MAPK Signaling Pathway ...... 152 Cell Cycle Dysregulation and Morphological Changes Occur with Arsenic Exposure ...... 156 Experimental...... 161 Results ...... 166 Intestinal UGT1A1 Induction Occurs with Oral Arsenic Exposure ...... 166 CAR is not Involved in Regulating Arsenic-Induced UGT1A1 Expression ...... 167 The NF-κB/IKK Pathway is not Involved in Regulating Intestinal UGT1A1...... 171 Oxidative Stress Induced Nrf2 Activation Upregulates UGT1A1 Expression...... 172 UGT1A1 Induction Occurs Independent of MAPK Activation ...... 175 Arsenic Exposure Causes Intestinal Damage, Changes in Cellular Morphology, and Increases Proliferation...... 176 Discussion...... 180 References ...... 188

CHAPTER 4 General Conclusions...... 202 Future Implications of this Work ...... 203 UGT Interactions in Human Hepatocytes ...... 203 Intestinal Microflora and UGT1A1 Induction ...... 206 References ...... 208

vii LIST OF ABBREVIATIONS

ADRs adverse drug reactions

AhR aryl hydrocarbon receptor

ARE antioxidant response element

ARNT aryl hydrocarbon receptor nuclear translocator

As3+ arsenite, inorganic trivalent arsenic

As5+ arsenate, inorganic pentavalent arsenic

B[a]P benzo[a]pyrene

CAR constitutive androstane receptor

CAT catalase

CN-I,II Crigler-Najjar syndrome type I or II

COX-2 cyclooxygenase-2

CPT-11 irinotecan

CYP cytochrome P450

DME drug metabolizing enzyme dsRNA double-stranded RNA

ER endoplasmic reticulum

ERK extracellular signal-related kinase

G6PD glucose-6-phosphate dehydrogenase

Gadd45β growth arrest and DNA-damage-inducible gene 45β

GI gastrointestinal tract

GPx glutathione peroxidase

GR glucocorticoid receptor

viii GSH-Re glutathione reductase

GST glutathione S-transferase

GSTA1,2 glutathione S-transferase A1 or 2

HCC hepatocellular carcinoma

H&E hematoxylin and eosin

HLM human liver microsomes

HO-1 heme oxygenase-1

HPLC-MS/MS high-performance liquid chromatography/tandem mass spectrometry

HRE hormone response element hUGT1 humanized UGT1 iAs inorganic arsenic

IKK IκB kinase

JNK c-Jun NH2-terminal protein kinase

Keap1 Kelch-like ECH-associated protein 1

Km Michaelis-Menten constant; substrate concentration at half the maximum rate of the reaction

Ki dissociation constant for an inhibitory enzyme-substrate complex

Ki-67 Ki-67 antigen

LPS lipopolysaccharide

LXRα liver X receptor α

MAPK mitogen-activated protein kinase

MC 3-methylcholanthrene

ix mEH microsomal epoxide hydrolase mRNA messenger RNA

NAC N-acetylcysteine

NAPQI N-acetyl-p-benzoquinone imine

NAT N-acetyltransferase

NF-κB Nuclear Factor κ B

Nrf2 NF-E2-related factor-2

NQO-1 NAD(P)H: quinone oxidoreductase 1

OPTI Only OPTI-MEM only was used in cell treatment

PAH polycyclic aromatic hydrocarbon

PAS Periodic Acid Schiff

PB phenobarbital

PBREM phenobarbital response enhancer module

PCN pregnenolone-16α-carbonitrile

PCNA proliferating cell nuclear antigen

PCR polymerase chain reaction

PPARα peroxisome proliferator-activated receptor

PXR pregnane X receptor

RISC RNA-Induced Silencing Complex

RNAi RNA interference

ROS reactive oxygen species

SI small intestine siRNA small interfering RNA

x SOD-1,2 superoxide dismutase-1 or -2

SULT sulfotransferase

TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin

TFF trefoil family factor peptide

TLC thin layer chromatography

Tg transgenic

TR Only the transfection reagent only was used in cell treatment

UDPGA uridine diphosphate glucuronic acid

UGT UDP-glucuronosyltransferase

Vmax the maximum rate of the reaction

XRE xenobiotic response element

xi LIST OF FIGURES

Figure 1-1. Organization of the human UGT1 locus...... 12

Figure 2-1. Glucuronidation of propofol in recombinant UGTs at therapeutically relevant concentrations...... 123

Figure 2-2. siRNA maps for the selected UGT1A9 and UGT2B7 oligos...... 124

Figure 2-3. siRNA down regulation of UGT1A9 and UGT2B7 in human hepatocytes ...... 125

Figure 2-4. Individual donor variation in siRNA down regulation was minimal for the three individual donors examined ...... 126

Figure 2-5. Inhibition of propofol glucuronidation by siRNA in human hepatocytes ...... 128

Figure 3-1. Effect of metal contaminants on serum bilirubin levels and UGT1A1 expression in neonatal hUGT1 mice...... 169

Figure 3-2. The CAR target gene Cyp2b10 is upregulated with arsenic exposure...... 170

Figure 3-3. CAR is not a direct regulator of arsenic-induced intestinal UGT1A1...... 170

Figure 3-4. Effects of arsenic exposure in IKK-α/IKK-β conditional knockout mice ...... 172

Figure 3-5. Arsenic exposure also induces Gsta1 and Gsta2 in the small intestine...... 174

xii Figure 3-6. Pre-treatment with NAC significantly reduces UGT1A1, Cyp2b10, and Gsta1 expression in small intestine ...... 174

Figure 3-7. Arsenic exposure does not activate JNK or ERK in intestine ...... 175

Figure 3-8. Tff1 is ectopically induced in small intestine with arsenic exposure...... 177

Figure 3-9. H&E staining reveals abnormal vacuole formation...... 179

Figure 3-10. PAS staining confirms abnormal vacuole formation...... 179

Figure 3-11. Increased PCNA concentrations and Ki-67 immunostaining confirm increased proliferation...... 180

Figure 3-12. The proposed mechanism for oral arsenic-induced UGT1A1 gene expression...... 187

xiii LIST OF TABLES

Table 2-1. Summary of the experimental conditions used for siRNA down regulation of UGT1A9 and UGT2B7 in human hepatocytes ...... 122

Table 2-2. Summary of kinetic parameters of propofol glucuronidation in human hepatocytes with and without siRNA treatment...... 122

xiv ACKNOWLEDGEMENTS

I would first like to thank my thesis advisor, Dr. Robert Tukey for the opportunity to work in his lab and do research in a field that I have always been fascinated by. His excitement for science has inspired me and his ability to clearly identify a story that needs be told is a quality I admire and strive to achieve. I am also thankful for his patience and confidence in me. He offered me direction when I needed it, but also encouraged me to trust in my own knowledge and abilities, which helped nurture me as an independent researcher. He has always had my best interest at heart and I am grateful for the invaluable advice and guidance, especially near the end of my graduate career. In addition, thank you for the opportunity of being your teaching assistant for Pharmacology and Toxicology. This experience has not only helped to solidify my understanding of material relevant to my research, but has allowed me to excel as a teacher and that is something in which I take great pride.

The Tukey Lab has been my family away from home for these last six years.

Each lab member has been integral in my development as a scientist and I have been very lucky to work with and learn from them. However, they have also made coming into work everyday something to look forward to and I am deeply grateful for their friendship. Dr. Shujuan Chen and Dr. Mei-Fei Yueh have both been very valuable resources to me. They were always willing to help and provided the best trouble- shooting advice. Thank you for your sweet personalities, unending kindness, and love of Dim Sum lunches. I thank Nghia Nguyen for providing me with invaluable technical expertise, but also appreciate his unconditional friendship. Deirdre Beaton

La Placa was instrumental in helping me develop my mouse-handling skills, but she

xv has played a very important role in developing my faith. Thank you for inspiring me with your own faith and supporting me during my journey towards my Catholic confirmation. Michelle Feiock and I attended several Superfund conferences together.

As a result, we became fast friends and I thank her for all the genuine laughs and extraordinary fun amidst the challenging graduate school environment. I would also like to acknowledge the past members of the lab. Erin Brace-Sinnokrak was very benevolent and kind to me when I first joined the lab. She helped keep me calm as I adjusted to the new world of graduate school and I thank her for her confidence, support, and friendship. Dr. Theresa Operaña’s intense dedication and passion towards science has been extremely inspiring. Thank you for teaching me to be vigilant and meticulous in my note-taking and lab work. Dr. Jessica Weems was incredibly supportive during the months leading up to my committee meeting and defense. Thank you for your genuine good nature and I enjoyed sharing my obsession with San Diego’s culinary gems with you. I would especially like to thank Dr.

Ryoichi Fujiwara. After he arrived from Japan to start his post-doctoral position in our lab, we quickly developed a strong alliance in the lab. While he helped keep me grounded and focused with his advice and positive reassurance, his most significant influence was teaching me how to ask the right questions. His work ethnic and scientific curiosity has been inspiring and he has helped me become a more confident, independent researcher. Most importantly, he has been a truly great friend and I am thankful for all the fun times in lab, at conferences, and on our gastronomic adventures during his time in San Diego.

xvi Additionally, the University of California, San Diego’s Chemistry &

Biochemistry Graduate Program has provided me with a stimulating and challenging environment in which to grow as a scientist. I would like to thank my thesis committee members: Dr. Pieter Dorrestein, Dr. James Halpert, Dr. Alexander

Hoffmann, and Dr. William Trogler for their guidance, helpful suggestions, and exciting scientific discussion. Additionally, Dr. Nissi Varki was an important resource in the UCSD Histology Core and I thank her for sharing her histology expertise with me. I would also like to acknowledge the Sacred Heart Community and Loyola

Marymount University, for being loving and nurturing environments that fostered both my creative and scientific spirit alike. You have helped me become the person I am today.

I am especially grateful to Robert Foti at Amgen, Inc. for giving me the epic opportunity to experience working in industry as a graduate intern in the PKDM department. This was one of the most formative experiences of my graduate career.

Rob continues to be an inspiring mentor and supportive ally and I am a better scientist after having worked with him. I would like to thank Dr. Leslie Dickmann for her help in the lab and her valuable input on our dimerization project, as well as Dr. Larry

Wienkers for being so generous with his time and guidance during my internship. I also could not have enjoyed my time at Amgen and in Seattle without the support of the other Amgen interns, especially Dr. Angela Bikker, Dr. Yan Wang, Robert Dong,

Kate Fan, and Rudy Nazitto. It was exhilarating being surrounding by such passionate and exceptional people. We absolutely grew as scientists together, but also left as

xvii lifelong friends, having shared in the remarkable opportunity of working at Amgen together.

Most importantly, I would like to thank my parents for instilling in me a great reverence for education. Their passion and dedication to knowledge and learning has and will continue to motivate me everyday. They also taught me very early on that anything and everything is possible, which was a very important message for a young, aspiring female scientist to hear growing up. I will never forget their endless encouragement and support, as they have been my unwavering cheerleaders in every aspect of my life. They have been instrumental in helping me establish a solid foundation upon which to build a future and I am forever grateful.

I would like to thank the rest of my family for providing me with the most wonderful, unending support as well. Their encouragement, celebration of accomplishments, and general attitude towards life has helped me throughout this challenging journey. I would especially like acknowledge my husband, Danny for encouraging me to challenge myself and inspiring me to strive for excellence, my cousin Alina for her amazing optimism, rationale and infinite support, my brother

Alek for his witty and refreshing comedic relief, my youngest cousin Olivia for keeping me grounded and being my reason for striving to be a positive, female role model, Ciocia Jay for reminding me to always question and think outside the box, and

Babcia for exemplifying what it means to be a strong female. To the rest of my family, friends, teachers, and students: “the light within me recognizes and honors the light within you”.

Chapter 2, in full, is a currently being prepared for submission in Archives of

xviii Biochemistry and Biophysics, 2012, Dickmann L., Tracy J., Tukey R.H., Wienkers

L.C., and Foti R.S. I was the primary investigator and author of this paper. Chapter 3, in part, is currently being prepared for submission for publication of the material. I was the primary investigator and author of this material.

xix VITA

EDUCATION 2006-2012 Ph.D. - Doctor of Philosophy (Chemistry and Biochemistry Graduate Program), University of California San Diego, La Jolla, CA

2006-2008 M.S. - Master of Science (Chemistry and Biochemistry Graduate Program), University of California, San Diego, La Jolla, CA

2001-2006 B.S. - Bachelor of Science (Chemistry and Biochemistry), Loyola Marymount University, Los Angeles, CA (Cum Laude)

RESEARCH EXPERIENCE Sep 2006-Sept 2012 Ph.D. Student: “UGT1A1 regulation by oral arsenic in humanized UGT1 mice”, supervised by Robert H. Tukey, Ph.D., Professor, Departments of Pharmacology and Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA.

Jun 2011-Sept 2011 Summer Research Intern: “Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on Propofol Glucuronidation in Human Hepatocytes”, supervised by Robert S. Foti, PKDM Scientist, Amgen, Inc., Seattle, WA.

TEACHING EXPERIENCE Mar 2008-Jun 2012 Teaching Assistant in undergraduate Pharmacology and Toxicology. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA.

Mar 2007-Dec 2007 Teaching Assistant in undergraduate Analytical Chemistry Laboratory. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA.

Jan 2007-Mar 2007 Teaching Assistant in undergraduate General Chemistry II. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA.

xx Sep 2006-Dec 2006 Teaching Assistant in undergraduate The Periodic Table. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA.

Aug 2004-May 2005 Teaching Assistant in undergraduate Organic Chemistry Laboratory. Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, CA.

Aug 2002-May 2004 Chemistry and Math Tutor at the Learning Resource Center. Loyola Marymount University, Los Angeles, CA.

HONORS AND AWARDS 2012 Teaching Assistant Excellence Award, Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA.

2005 Academically Outstanding Graduating Senior in Chemistry, Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, CA.

2005 President of the Chemistry Society, Loyola Marymount University chapter of the American Chemical Society.

2001-2005 Academic Dean’s List, Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, CA.

PUBLICATIONS Konopnicki C.M., Dickmann L., Tracy J., Tukey R.H., Wienkers L.C., and Foti R.S. Evaluation of UGT Protein Interactions in Human Hepatocytes: Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on Propofol Glucuronidation in Human Hepatocytes.

ABSTRACTS AND PRESENTATIONS 1. “Using a P450 HRGS assay to test complex environmental toxicant mixtures along the U.S.-Mexico border” at the Superfund Basic Research Program Annual Meeting in Raleigh Durham, North Carolina, December 2007.

xxi 2. “Serum Bilirubin Levels in Humanized UGT1A1*28 Mice Can Serve as a Marker for Toxicant Exposure” at the Superfund Basic Research Program Annual Meeting in Asilomar, California, December 2008.

3. Serum Bilirubin Levels in Humanized UGT1A1*28 Mice Can Serve as a Marker for Toxicant Exposure” at UCSD-Pfizer Global Research and Development (DMPK Symposium), San Diego, La Jolla, California, September 2009.

4. “Arsenic Exposure Modulates UGT1A1 Expression in Humanized UGT1 Mice” at the Superfund Basic Research Program Annual Meeting in New York City, New York, November 2009.

5. “Serum Bilirubin Levels in Humanized UGT1A1*28 Mice Can Serve as a Marker for Heavy Metal Exposure” at the UCSD-Pfizer Global Research and Development (DMPK Symposium), San Diego, La Jolla, California, October 2010.

6. “Serum Bilirubin Levels in Humanized UGT1A1*28 Mice Can Serve as a Marker for Heavy Metal Exposure” at the Superfund Basic Research Program Annual Meeting in Portland, Oregon, November 2010.

7. “The Regulatory Role of Oral Arsenic in Humanized UGT1 Mice” at Pharmacology Research Discussions, San Diego, La Jolla, California, January 2011.

8. “siRNA Knockdown of UDP-Glucuronosyltransferases in Human Hepatocytes” at the Amgen Summer Intern Program Poster Session, Seattle, Washington, August 2011.

9. “siRNA Knockdown of UDP-Glucuronosyltransferases in Human Hepatocytes” at PKDM Department Meeting at Amgen, Seattle, Washington, August 2011.

10. “siRNA Knockdown of UDP-Glucuronosyltransferases in Human Hepatocytes” at the Superfund Basic Research Program Annual Meeting in Lexington, Kentucky, October 2011.

11. “Oral arsenic exposure induces UGT1A1 expression in neonatal humanized UDP- Glucuronosyltransferase-1 mice through changes in cellular morphology associated cytotoxicity” at the Experimental Biology: ASPET Meeting in San Diego, California, April 2012.

12. “Oral arsenic exposure induces UGT1A1 expression in neonatal humanized UDP- Glucuronosyltransferase-1 mice through changes in cellular morphology associated cytotoxicity” at the SCDMDG Meeting, San Diego, La Jolla, California, October 2012.

xxii ABSTRACT OF THE DISSERTATION

The Intricacies of UGT Regulation:

Protein-Protein Interactions and Environmental Arsenic Exposure

Camille Maria Konopnicki

Doctor of Philosophy in Chemistry

University of California, San Diego, 2012

Professor Robert H. Tukey, Chair

The human UDP-glucuronsyltransferases (UGTs) are responsible for the metabolism of many endogenous and exogenous compounds. They facilitate excretion by attaching glucuronic acid to a lipophilic parent compound, transforming it to a more water-soluble glucuronide that can be easily eliminated.

Experiments performed in recombinant systems have suggested that protein- protein interactions occur between the UGTs and may play a role in modulating activity. However, evidence of UGT interactions either in vivo or in more

xxiii physiologically relevant in vitro systems has yet to be demonstrated. UGT oligomerization and its ability to affect glucuronidation were examined by siRNA knockdown and activity studies. Selective down regulation of UGT1A9 or UGT2B7 resulted in significant decreases in their respective mRNA levels. As expected, metabolism of the UGT1A9 probe substrate propofol was abolished with UGT1A9 down regulation. UGT1A9 activity also decreased with UGT2B7 down regulation, implying potential interactions between two isoforms. This represents the first piece of evidence that UGT interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact glucuronidation of selective

UGT1A9 substrates.

UGT1A1 is one of the most important UGTs because it is the primary UGT responsible for bilirubin metabolism. The UGT1A1 gene is also regulated by almost all of the xenobiotic receptors. We have recently generated a humanized UGT1 mouse model that exhibits elevated bilirubin levels during development. Since numerous toxicants induce UGT1A1 through association with xenobiotic receptors, UGT1A1 induction by environmental contaminants can alter hUGT1 bilirubin levels, therefore serving as a sensor for toxicant exposure. We investigated association between prominent metal contaminant exposure and UGT1A1 expression through fluctuations in bilirubin. Arsenic exposure reduced bilirubin in neonatal hUGT1 mice and significantly induced intestinal UGT1A1. The prevalence of arsenic contamination throughout the world fueled investigation into the regulatory role of oral arsenic on

UGT1A1 expression. Q-PCR, Western Blot, and immunohistological analysis revealed a novel mechanism that implicates Nrf2, NF-κB and cellular proliferation as

xxiv potential underlying regulators of arsenic-induced UGT1A1 expression in hUGT1 mice.

xxv

CHAPTER 1

Introduction to Drug Metabolism

1 2

Drug Metabolizing Enzymes from an Evolutionary Perspective

While drug metabolizing enzyme (DME) genes have existed on this planet for more than 2.5 billion years, their evolutionary purpose has not solely been for what we associate these enzymes with today – the clearance of drugs. DMEs are evolutionarily very old enzymes and it is understood that they first evolved to serve critical life functions in both prokaryotes and eukaryotes (Nebert and Dieter, 2000). These enzymes have evolved to cope with selective pressures such as adaptability, environmental impact, and diet. What we have come to know as protective processes associated with drug clearance are actually natural mechanisms to provide the body with nutrients and dispose of unwanted material. The theory that life evolved from a

“primordial soup” of limited organic compounds provides some of the earliest evidence of selective pressure, requiring evolution of biosynthetic pathways in order to replace dwindling supplies (Alves et al., 2002). The anaerobic conditions of the earth’s environment have led researchers to believe that some of the earliest

Cytochrome P450s (P450s) performed vital reductase and isomerase functions

(Lechner, 1994). Later, at the advent of an oxygenated environment, mechanisms to detoxify oxygen and protect against oxidative stress became necessary for species survival (Sistonen et al., 2009). The existence of numerous DMEs, even prior to the divergence of prokaryotes and eukaryotes, highlights their importance for many life sustaining processes, including membrane synthesis, calcium ion and electrolyte balance, cell division, development, and the metabolism of endogenous substrates

(Nebert and Dieter, 2000).

Evolutionary divergence of DME genes in animals over the last 1,000

megaannum has been strongly influenced by necessary interactions between animals and plants at the emergence of terrestrial life (Coveney et al., 2012). While plants required animals for their reproductive cycles, they also needed ways to protect themselves from being consumed. As a means of defense, plants evolved new genes and thus metabolites, such as phytoalexins (Bock, 2003), to make them less enticing or even toxic, which forced animals to evolve new DME genes to adapt to the constantly changing plants (Gonzalez and Nebert, 1990). Animal consumption of available plant life launched this co-evolutionary event known as “animal-plant warfare” and resulted in an explosion of animal DME gene duplication events, as a means to cope with new dietary constituents. The role of DMEs has more recently expanded to include metabolic bioactivation and detoxification of numerous environmental pollutants, carcinogens, and drugs that are now found in humans (Nebert, 1997). The development of defense mechanisms to detoxify plant metabolites by animal DMEs undoubtedly played a significant role in our current ability to detoxify such a vast array of substrates. As the pace of the chemical revolution has overtaken biological evolution, the prominent role of DMEs in the clearance of man-made substrates, including drugs, pesticides, carcinogens and other xenobiotics, has become apparent

(Burchell and Coughtrie, 1989).

The Function of Phase I and Phase II Drug Metabolizing Enzymes

DMEs assist in the metabolism, detoxification, and elimination of many endogenous compounds, as well as chemical agents that are not dietary in origin – classified as “xenobiotics”. Many of these compounds are lipophilic to enable

diffusion through membranes to effector sites and processes to render them more water-soluble are needed to facilitate their removal from cells and the organism as a whole. The elimination of many lipophilic xenobiotic compounds can be divided into two distinct but closely linked systems: Phase I and Phase II metabolism (Williams,

1949). Phase I oxidative metabolism by cytochrome P450 enzymes is the primary method of drug metabolism. Phase I metabolism involves small molecule modifications such as oxidation, hydroxylation, peroxidation, and dealkylation that introduce functional groups to the substrate (Nebert, 2006; Guillemette, 2003). This functionalization step is often, but not always, required for subsequent conjugative

Phase II metabolism to occur. Phase II enzymes utilize these reactive groups to covalently attach large, polar moieties, such as glucuronic acid, sulfate, and glutathione. The result is a more polar, conjugated metabolite that can easily be eliminated from the body (Williams, 1971; Omiecinski et al., 2011). It is important to note that Phase I and Phase II metabolism can also result in active and/or toxic metabolites that must be further detoxified.

Characterization of the Glucuronidation Process

Endogenous and exogenous compounds are detoxified and eliminated from the body through concerted metabolism between Phase I and Phase II DMEs. Phase II metabolism is characterized by the conjugation of large, hydrophilic groups to reactive moieties, thus increasing the molecular weight and water solubility of the substrate.

The UDP-glucuronosyltransferases (UGTs) are Phase II enzymes that catalyze the addition of the bulky sugar group, glucuronic acid, to a nucleophilic substrate, termed

the “aglycone”, utilizing UDP-glucuronic acid (UDPGA) as a co-substrate. This detoxification process is called glucuronidation and the resulting sugar conjugate is referred to as a glucuronide. Glucuronidation is the major pathway in Phase II metabolism and accounts for approximately 35% of drug conjugation, making the

UGTs the most important Phase II enzymes for the detoxification of drugs

(Guillemette, 2003).

The first compound characterized as a sugar conjugate was euxanthic acid. It was isolated in 1855, by German scientist W. Schmidt, who fed mango leaves to cows and isolated compounds from their urine (Dutton, 1966; Dutton, 1980). Throughout the 1870s, other sugar containing metabolites were isolated from urine in various drug metabolism studies (Conti and Bickel, 1977). In 1879, Schmiedeberg and Meyer were the first to isolate and characterize the sugar moiety, glucuronic acid, while studying camphor metabolism in dogs (Schmiedeberg and Meyer, 1879; Pryde and Williams,

1933). Lipschitz and Bueding demonstrated in 1939 that the major site of glucuronide production was the liver (Lipschitz and Bueding, 1939). However, very little else was known at the time about the mechanisms by which glucuronic acid was synthesized within the body or how it became incorporated into those compounds that contained it

(Dutton and Storey, 1953). The year 1953 marked a milestone discovery for Dutton and Storey, who had discovered a thermostable cofactor in the liver that was required for phenol glucuronide formation in cell-free preparations (Dutton and Storey, 1954).

Further characterization determined this compound to be UDPGA, the active cofactor required in the reaction to form glucuronide metabolites (Storey and Dutton, 1955) and that the transfer of glucuronic acid to a substrate occurred by the following

general mechanism: UDP Glucuronic acid + R-OH  UDP + R-O-Glucuronic acid, where UDP-Glucuronic acid, R-OH, UDP and R-O-Glucuronic acid represent the factor, substrate, uridine diphosphate, and the glucuronide, respectively (Smith and

Mills, 1954).

UDP-Glucuronsyltransferases

In 1957, it was confirmed that all UGT substrates possess a nucleophilic functional group (Axelrod et al., 1957). The wide range of substrates capable of being glucuronidated led to deliberations over the existence of a single UGT enzyme with a promiscuous active site (Mulder, 1971) or a population of numerous transferases, each with its own specific substrate (Storey, 1965; Gram et al., 1968; Temple et al., 1968).

Early purification of the UGTs was challenging, due to the relative instability of UGTs in high concentrations of detergent required for solubilization and their phospholipid dependence (Burchell and Coughtrie, 1989). Without a method to isolate these enzymes from animal tissues, it was impossible to further address the question of heterogeneity. In 1975, Del Villar et al. demonstrated successful UGT purification by

DEAE-cellulose column chromatography and confirmed that morphine and p- nitrophenol were conjugated by separate enzymes. This was the first evidence demonstrating that glucuronsyltransferase activity was attributable to more than one enzyme (Del Villar et al., 1975). Also, through the use of DEAE-cellulose column chromatography, Bock et al. purified and separated two rat liver UGTs that catalyzed either morphine or 1-napththol and morphine glucuronidation (Bock et al., 1977). In

1977, Gorski and Kasper published a method for purifying UGTs to homogeneity that

utilized affinity chromatography with UDP-hexanolamine Sepharose. The isolated enzymes had much higher specific activity (Gorski and Kasper, 1977) and several

UGTs were subsequently purified by this method, including phenobarbital-inducible

UGT (Burchell, 1978), oestrone-UGT and p-nitrophenol-UGT (Tukey et al., 1978), and testosterone-UGT (Weatherill and Burchell, 1980). Additional findings from these purification experiments demonstrated that the catalytic activity of many membrane-bound enzymes was dependent upon the presence of phospholipids, which also validated earlier reports of UGT localization within the endoplasmic reticulum membrane. Tukey et al. demonstrated that partially purified, delipidated enzymes were essentially catalytically inactive, yet activities could be restored upon addition of phospholipids or phosphatidylcholine mixtures (Tukey and Tephly, 1980; Gorski and

Kasper, 1977; Tukey et al., 1978).

With a reliable purification method, the UGT field evolved as further characterization of purified isoforms became possible. Substrate specificity analysis revealed that the UGTs displayed distinct and overlapping substrate specificities, thus strengthening the proposal that the functional heterogeneity was due to heterogeneous population of UGTs (Falany and Tephly, 1983; Roy Chowdhury et al., 1986). To determine homology between purified isoforms, physical characterization was assessed through peptide mapping and amino acid analysis. In 1986, Falany et al. observed that the 17-hydroxysteroid-UGT and 3-hydroxyandrogen-UGT displayed significant homology, despite their different substrate specificities and that the p- nitrophenol-UGT was less closely related to the two steroid UGTs as it displayed very different amino acid composition and peptide mapping (Falany et al., 1986). These

findings suggested the possibility that families of related UGTs existed and was later confirmed through comparison of nucleotide sequences of cloned cDNAs encoding for

UGT isoforms (Jackson and Burchell, 1986; Mackenzie, 1986; Mackenzie, 1987;

Harding et al., 1987; Iyanagi et al., 1986).

Advancements in homogenous UGT isolation eventually led to the production of antibodies raised against purified UGTs, which could be used to specifically identify and immunoprecipitate nascent UGT proteins translating from polysomes

(Mackenzie et al., 1984a). The first mouse UGT mRNAs were isolated utilizing this technique, in addition to several of the rat liver UGTs (Mackenzie et al., 1984b;

Jackson et al., 1985). Comparison of mRNA sequences suggested the existence multiple UGT isoforms within at least two gene families, but most importantly, directly confirmed the heterogeneity of UGTs. This method led to a significant increase in isolation and characterization of UGTs from various species including rat, mouse, and human, ultimately resulting in the development of guidelines for standardized nomenclature based on evolutionary divergence (Burchell et al., 1991;

Mackenzie et al., 1997).

The UGTs have been divided into two separate gene families, UGT1 and

UGT2, on the basis of their sequence homology (Burchell et al., 1991; Mackenzie et al., 1997). The UGT2 family is composed of individual genes, each consisting of a promoter and 6 exons, clustered on chromosome 4 at 4q13-q21. This gene cluster encodes 7 functional UGT2B proteins, as well as 3 UGT2A proteins that have yet to be functionally characterized (Monaghan et al., 1994; Beaulieu et al., 1997; Chen et al., 1993). The UGT2B enzymes specifically catalyze the glucuronidation of bile

acids (Monaghan et al., 1997), steroids (Belanger et al., 1998; Hum et al., 1999), and hormones (Jin et al., 1997; Guillemette et al., 2004; Thibaudeau et al., 2006), but have also been identified in the elimination of some therapeutic agents (Yeh, 1975;

Coffman et al., 1997; Davies et al., 2003).

The UGT1 gene family is located on chromosome 2 at 2q37 (Harding et al.,

1990). The UGT1 enzymes are predominantly involved in the metabolism of exogenous compounds (Dutton, 1980) with the important exception of the endogenous compound, bilirubin. Bilirubin is the yellow breakdown product of hemoglobin and if left unbound, can potentially lead to severe hyperbilirubinemia that ultimately results in brain damage due to excessive accumulation of bilirubin in the brain (Crigler and

Najjar, 1952a; Gourley, 1997). It had already been established in 1956 that conjugation to form the glucuronide was the only method of bilirubin clearance

(Billing et al., 1957; Schmid, 1956; Talafant, 1956). However, in 1991, Ritter et al. identified two UGTs that displayed activity for bilirubin, HUG-Brl and HUG-Br2

(Ritter et al., 1991). Bosma et al. later amended these findings, having determined that solely one isoform, UGT1A1, was capable of efficiently conjugating bilirubin

(Bosma et al., 1994). Sequence data, from studies also performed by Ritter and his co-workers during their search for the gene coding for the bilirubin transferases, revealed that the HUG-Brl and HUG-Br2 cDNAs contained 3’ ends identical to each other, as well as to the human phenol transferase cDNA, HLUG P1. These results coincidentally provided the first evidence of a novel gene locus encoding for the

UGT1 family. The organization and structure of the gene complex was determined to utilize a series of unique exon 1s with accompanying transcriptional start sites and

differential splicing to commonly shared exons to generate six different mature mRNAs (Ritter et al., 1992). The UGT1 complex was later extended to 220 kb and determined to encode for a total of 13 isoforms: four psuedogenes and nine functional isoforms (Gong et al., 2001). These nine UGTs are generated through the transcription process of exon sharing, where exon 1s, which appear to have evolved through gene duplication events (Mackenzie et al., 2005), are spliced and joined to common exons 2 through 5 (Figure 1-1). The resulting UGT1A proteins therefore have a variable N-terminal domain of approximately 280 amino acids, and an identical, 245-amino acid, C-terminal domain (Wooster et al., 1991). Although the

UGT1A proteins are encoded by five exons and UGT2 proteins by six exons, the carboxyl termini are highly conserved between all UGTs. It has been established that the variable N-terminal region (exon 1 for UGT1A; exon 1-2 for UGT2) dictates isoform substrate specificity (Mackenzie, 1990), while the highly conserved C- terminal region (exons 2-5 for UGT1A; exons 3-6 for UGT2) contains the co-substrate binding site (Wooster et al., 1993; Burchell and Coughtrie, 1989; Ritter et al., 1992;

Tephly and Burchell, 1990; Radominska-Pandya et al., 1999).

The ability to further study individual isoforms from complex gene families, such as the UGT1 family, was achieved through the application of modern molecular biological techniques. Cloning of available UGT cDNAs encoding a single enzyme and subsequent transient or stable transfection into cell types with low levels of target enzymes generated in vitro models heterologously expressing individual enzymes that facilitated the characterization of individual UGT1A gene products (Mackenzie, 1986;

Remmel and Burchell, 1993; Wooster et al., 1993; Strassburg et al., 1996; Strassburg

et al., 1998; Strassburg et al., 1999a; Remmel et al., 2009). Recombinant protein harvested from cells could be incubated with specific substrates in the presence of radiolabeled UDPGA to assess UGT enzymatic activity via thin layer chromatography

(Nguyen and Tukey, 1997). Characterization through cDNA expression experiments led to the identification of more than 350 individual compounds that serve as substrates for the UGT superfamily (Tukey and Strassburg, 2000). This large group of structurally divergent compounds covers many different chemical classes, including alcohols, flavones, coumarins, carboxylic acids, amines, opioids, and steroids (Tukey and Strassburg, 2001). Additionally, many daily dietary constituents and pharmaceutical drugs contain the same reactive groups associated with these classes of agents, making them ideal substrates for glucuronidation.

Figure 1-1. Organization of the human UGT1 locus. The UGT1 locus is located on chromosome 2 and spans 220 kb. The locus contains 13 unique exon 1 cassettes that can be spliced and joined to common exons 2-5. The nine functional UGT1A transcripts are generated through this transcription process of exon sharing. The 3’ splice site of exon 1 is directly spliced to the 5’ splice site of conserved exon 2. Due to absence of splices sites on their 5’ ends, all intervening exons are considered intronic and spliced out. The presence of unique TATA-like elements approximately 30 bp upstream of each functional exon 1 allows for individual transcriptional regulation.

Tissue-Specific Expression of the UGTs

Human glucuronidation studies have primarily focused on hepatic tissue, because of the greater availability of the tissue source and the well-understood role of the liver in drug metabolism. However, the diverse nature of substrate specificity displayed by the UGT1 enzymes suggests that the existence of their complex gene regulation is most likely designed to account for the variable and specific glucuronidation requirements in various organs (Strassburg et al., 1997b). Several studies have documented UGT activity toward bile acids, phenols, and bilirubin in human intestinal (Matern et al., 1984; Pacifici et al., 1986; Parquet et al., 1985; Peters

et al., 1989; Peters et al., 1991; McDonnell et al., 1996) and renal tissues (Parquet et al., 1985; Peters et al., 1989; Pacifici et al., 1988; Peters et al., 1987; Peters and

Jansen, 1988). It is now clear that human UGT1 gene expression is regulated in a strict tissue-specific manner, resulting in varying levels and complementations of

UGT1A proteins in each tissue (Strassburg et al., 1997b; Tukey and Strassburg, 2000;

Dutton, 1978). The use of RT-PCR, a method by which it was possible to distinguish single base pair differences between the highly homologous UGT sequences, advanced the study of tissue-specific expression patterns and provided a way to semi- quantify relative mRNA abundance of UGT transcripts within tissues (Strassburg et al., 1997a; Strassburg et al., 1997b). Expression of 17 UGT enzymes has been observed in humans. These include the nine functional UGT1A isoforms (UGT1A1,

UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9,

UGT1A10), seven UGT2Bs (UGT2B4, UGT2B7, UGT2B11, UGT2B15, UGT2B17,

UGT2B28), and one UGT2A enzyme (UGT2A1) (Tukey and Strassburg, 2000).

Isoforms found to be expressed in liver are UGT1A1, UGT1A3, UGT1A4, UGT1A6, and UGT1A9 (Strassburg et al., 1997a). UGT1A7, UGT1A8, and UGT1A10, which are notably absent in liver, are exclusively expressed extrahepatically (Strassburg et al., 1997b; Strassburg et al., 1998). UGT1A8 and UGT1A10 have been identified throughout the gastrointestinal (GI) tract, predominantly in the small intestine and colon, whereas UGT1A7 expression is limited to the upper GI tract, including the esophagus and stomach (Vogel et al., 2002; Zheng et al., 2002; Strassburg et al.,

1999b). Examination of non-GI extrahepatic tissues revealed that UGT1A7 was also expressed in pancreas (Ockenga et al., 2003), that UGT1A9 was expressed in kidney

(McGurk et al., 1998), and UGT1A6 in rat (Suleman et al., 1998) and human (King et al., 1999) brain. The colon exhibits an abundance of UGT1As, with UGT1A7 being the only isoform not detected (Strassburg et al., 1998). UGT2 gene expression also occurs in a tissue-specific manner. With the exception of UGT2B17, hepatic expression of the other six isoforms has been identified in human liver (Jin et al.,

1993; Beaulieu et al., 1998; Strassburg et al., 1999b; Green et al., 1994; Jackson et al.,

1987; Chen et al., 1993). UGT2B10, UGT2B11, UGT2B15, and UGT2B17 expression has been observed in steroid-sensitive tissues, including the prostate and mammary glands, as well as throughout the GI tract (Tukey and Strassburg, 2000;

Beaulieu et al., 1996). UGT2B11 exhibits the most extensive expression, having been identified in liver, kidney, breast, prostate, skin, adipose tissue, adrenal tissue, and lung (Beaulieu et al., 1998). UGT2A1 expression is particularly tightly regulated and has been distinctly restricted to olfactory tissue. It is believed that UGT2A1 plays a role in human odorant sensing and also serves as a first line of metabolic defense for airborne toxic compounds entering the body through the nasal passage (Jedlitschky et al., 1999). It is not surprising that strict tissue-specific regulation of DMEs ensures proper clearance of xenobiotics as tissues become exposed to them (Tukey and

Strassburg, 2001; Strassburg et al., 1999a).

Individual UGT Variability, Disease, and Adverse Drug Reactions

Individual variability in DMEs, due to environment, lifestyle and genetic influences, plays a significant role in the xenobiotic response (Burchell et al., 2000).

Disruption of natural metabolic processes most commonly occurs through

environmental exposures, which can cause xenobiotic-, toxicant-, or dietary-initiated enzyme induction or inhibition. Enhancement or reduction in gene expression of important enzymes can ultimately lead to altered biotransformation, causing imbalances in endogenous compounds or adverse drug reactions (ADRs). In 1999,

Ritter et al. studied UGT1A1 variation in human donor livers and their corresponding primary hepatocyte cultures. The three donors with the highest UGT1A1 levels had a history of phenytoin exposure. Phenytoin is an anticonvulsant and known UGT inducer and these findings were consistent with clinical evidence demonstrating the effectiveness of PB in inducing bilirubin clearance in patients with hyperbilirubinemia. Prototypical inducing agents, PB, phenytoin, and oltipraz also elevated UGT1A1 mRNA in isolated primary hepatocytes. However, the most significant induction was seen with 3-methylcholanthrene (MC), illustrating that exposure to polycyclic aromatic hydrocarbons (PAHs), which are potent atmospheric pollutants found in cigarette smoke, can effect UGT1A1 variation (Ritter et al., 1999;

Wells et al., 2004).

In many cases, a decrease in expression of a specific UGT results in a compound being diverted to an unfavorable pathway that will convert it to a reactive metabolite capable of covalently binding DNA and proteins. This is known to occur with the over-the-counter pain reliever, acetaminophen (Tylenol). Glucuronidation is the major route of acetaminophen clearance and therefore reduced UGT activity leads to its accumulation. Inability to clear acetaminophen by its primary pathway diverts elimination through one of the minor elimination routes, oxidation by the CYPs, resulting in the formation of the toxic intermediate, N-acetyl-p-benzoquinone imine

(NAPQI). At normal acetaminophen doses, NAPQI can be easily detoxified by glutathione; however, glutathione becomes severely depleted at toxic levels. This leaves NAPQI free to bind protein thiols, which can affect protein activity, ultimately resulting in acute liver failure and severe renal damage (Hinson et al., 1980; Uetrecht,

2010). Studies performed by de Morais et al. have shown that Gunn rats, which are inherently deficient in UGT1A proteins, are more susceptible to acetaminophen toxicity in comparison to normal Wistar controls, thus supporting adverse biological outcomes in the absence of UGT1A enzymes (de Morais et al., 1992).

UGT1A1 plays a particularly important role in the glucuronidation of a wide array of compounds, the most notable being bilirubin. Bilirubin is an endogenous byproduct of heme derived from hemoglobin, cytochromes, catalase, peroxidase, other hemoproteins, and a small pool of free heme (Tenhunen et al., 1969). It is known for its inherent antioxidant properties at low concentrations and has even been proven to decrease cardiovascular disease risk in patients with low UGT1A1 levels (Melton et al., 2011). In the blood circulation, bilirubin is bound to albumin in order to prevent toxicity caused by the unbound form. Despite high-affinity binding to albumin, bilirubin is rapidly and selectively taken up from the blood circulation into the liver

(Cui et al., 2001), where it is conjugated by UGT1A1 to form mono- and di- glucuronides. Defects in clearance of bilirubin lead to accumulation of unsafe levels of unbound bilirubin (≥ 20 mg/dL) and are associated with serious toxicities, including

CNS toxicity, brain damage (kernicterus), and even death (Fujiwara et al., 2010b;

Ritter et al., 1999). Therefore, control of bilirubin levels in the body is absolutely critical.

There are three forms of inheritable unconjugated hyperbilirubinemia diseases that occur in humans: Crigler-Najjar syndrome type I (CN-I), Crigler-Najjar syndrome type II (CN-II), and Gilbert’s syndrome (Kadakol et al., 2000). CN-I was first described by Crigler and Najjar in 1952 and is characterized by potentially lethal hyperbilirubinemia due to severely high serum bilirubin levels that range from 20–50 mg/dL (Crigler and Najjar, 1952a; Crigler and Najjar, 1952b). The severity of CN-I was later determined to result from the complete absence of UGT1A1 protein, and therefore activity (Seppen et al., 1994). While phototherapy treatment has extended life expectancy, it eventually becomes less effective. Liver transplantation is currently the only restorative method available for patients with CN-I (Gourley, 1997). CN-II is characterized by intermediate hyperbilirubinemia (~7-20 mg/dL) due to decreased

UGT1A1 activity (Arias, 1962; Seppen et al., 1994). Induction therapy with PB to induce residual enzyme activity has been used to treat CN-II patients (Arias et al.,

1969). CN-I and CN-II syndromes both result from mutations in any of the five exons of the UGT1A1 gene. Mutations that cause a premature stop codon or shift the reading frame ultimately alter or delete a large number of amino acid residues and always result in abolished UGT1A1 activity as seen in CN-I (Jansen et al., 1995; Kadakol et al., 2000; Bosma et al., 1993; Ciotti et al., 1998). In contrast, Gilbert’s syndrome is a relatively harmless, commonly inherited condition characterized by mild hyperbilirubinemia (Kadakol et al., 2000) and 3 to 10 percent of the general population is predicted to have it (Owens and Evans, 1975; Bailey et al., 1977; Sieg et al., 1987). The observed reduced UGT1A1 activity is due to an extra TA insertion in the TATA box of the UGT1A1 promoter (A(TA)6TAA is normal), which results in

decreased promoter activity. Bosma et al. documented a 70% decrease in UGT1A1 transcription in Gilbert’s syndrome patients (Bosma et al., 1995).

Characterization of UGT1 polymorphisms has led to the identification of certain UGT gene variants as risk factors for cancer (Guillemette et al., 2000a).

Inherited variations in genes involved in the metabolism of estrogens have been implicated in the increased risk of breast cancer. Estrogens are essential for development of the reproductive system in women. However, estrogen exposure for lengthy periods of time may cause breast cancer since prolonged proliferation and genetic instability in estrogen target tissues has been thought to increase the likelihood of normal cells transforming into a malignant type (Guillemette et al., 2004).

UGT1A1 glucuronidation directly inactivates estrogens to facilitate their removal from estrogen-sensitive tissues. Investigation of the association between genetic variability in the UGT1A1 promoter region and estrogen-related cancer risk revealed that reduced levels of UGT1A1 increased breast cancer susceptibility (Guillemette et al., 2000a).

Many cancer-causing environmental contaminants, such as PAHs found in cigarette smoke are detoxified by glucuronidation (Bock et al., 1999). Since UGT efficiency is critical for toxicity protection, polymorphic variation in the UGTs has the potential to alter sensitivity to PAHs present in diet and the environment (Hu and Wells, 1994).

Enhanced DNA adduct formation has been observed in UGT1A deficient Gunn rats exposed to the PAH, benzo[a]pyrene (B[a]P) (Hu and Wells, 1992). UGT1A7 is an important extrahepatic UGT expressed in orolaryngeal tissues and lung. These tissues are in direct contact with cigarette smoke and therefore it is not surprising that

UGT1A7 is responsible for the detoxification of several tobacco carcinogens

(Guillemette et al., 2000b). When Zheng et al. examined the potential role of

UGT1A7 genotype in orolaryngeal cancer risk, it was observed that UGT1A7 allelic variants resulting in low enzyme activity were associated with increased risk of head and neck cancer (Zheng et al., 2001). UGT1A7 has also been implicated as a cancer risk gene for liver and colon cancer (Vogel et al., 2001; Strassburg et al., 2002).

While UGT1 deficiencies have been studied with respect to disease susceptibility, a more immediate impact has recently emerged concerning treatment with drugs that have narrow therapeutic indices as well as more commonly used drugs with the potential for unwanted side effects. It is believed that polymorphisms within genes encoding for the UGTs can significantly affect drug response; therefore, patients with glucuronidation deficiencies are at a potentially higher risk for drug toxicities even when given normal therapeutic doses (Radu and Atsmon, 2001). The anticancer drug, irinotecan (CPT-11) is a classic example of pharmacogenetic influence on drug disposition and response. CPT-11 is hydrolyzed to its active metabolite SN-38, which is mainly eliminated through glucuronidation by UGT1A1. Individuals with Gilbert’s syndrome that exhibit decreased enzyme activity have been determined to be at greater risk of irinotecan-induced toxicity (Wasserman et al., 1997). Gilbert’s syndrome patients are also susceptible to adverse drug reactions (ADRs) with the protease inhibitor atazanavir (Lankisch et al., 2006) as well as a number of common therapeutic drugs, including acetaminophen (Douglas et al., 1978) and ibuprofen (Radu and

Atsmon, 2001). These findings support the effect of UGT genetic variation on individual sensitivities toward various therapeutic agents (Wells et al., 2004). The future of pharmacy is geared toward understanding inherited differences in drug

disposition or drug effects in hopes of improving drug safety and establishing personalized pharmacotherapy, which would involve optimized drug therapy based on a patient’s unique genetic makeup (Strassburg, 2008).

Protein-Protein Interactions Between DMEs

Drug oxidation and conjugation by CYPs and UGTs, historically, have been considered to occur separately. However, recent studies have suggested that protein- protein interactions occur between DMEs and can even facilitate enzyme efficiency and function. Classic drug metabolism is typically understood as steps in which a polar moiety is introduced into a compound so that it will be more suitable for excretion. Although CYP metabolism generally leads to less active and more polar metabolites, bioactivation of pro-drugs and pro-carcinogens can result in pharmacologically or toxicologically active substances, respectively (Vandenbrink et al., 2012; Ishii et al., 2010). The UGTs and other Phase II enzymes, such as N- acetyltransferases (NATs), sulfotransferases (SULTs), and glutathione S-transferases

(GSTs), play an important role in detoxifying these potent carcinogenic metabolites

(Tukey and Strassburg, 2000; Operaña and Tukey, 2007; Ishii et al., 2005). It would be reasonable to expect that a reactive metabolite produced by the CYPs could be directly transferred to other enzymes participating in its metabolism via protein- protein interactions, as physical interactions between DMEs would allow for the most efficient, concerted metabolism and aid in minimizing toxicity (Takeda et al., 2005;

Srivastava and Bernhard, 1987).

Increasing evidence of DME interactions has served to further validate that

enzymes act in a cooperative manner to metabolize xenobiotics rapidly and efficiently

(Taura et al., 2000). In 2000, Taura et al. used affinity chromatography to demonstrate that CYP1A1 is associated with microsomal epoxide hydrolase (mEH),

UGTs, and NADPH cytochrome P450-reductase (Taura et al., 2000). Following in

2005, Fremont et al. investigated the immunoprecipitation of several human UGT isoforms and CYP3A4 in human liver microsomes by P450-immobilized affinity chromatography. These studies demonstrated successful co-immunoprecipitation of

CYP3A4 with UGT2B7, UGT1A1, and UGT1A6 (Fremont et al., 2005). With ample research suggesting CYP-UGT associations, it was important to address their potential functional relevance. Co-expression of CYP3A4 and UGT2B7 in COS cells was found to greatly increase UGT2B7-catalyzed glucuronidation of morphine, providing some of the earliest evidence of these postulated cooperative interactions in modulating enzyme function (Takeda et al., 2005). However, another study found there to be no effect on rat UGT1A6 activity when it was simultaneously expressed with rat CYP1A1, suggesting that CYP-UGT interactions might actually be isoform specific (Ikushiro et al., 2004; Takeda et al., 2005; Ishii et al., 2005). Characterization of isoform specificity of CYP-UGT interactions was tested via co- immunoprecipitation experiments, where CYP isoform-specific antibodies were used as probes to co-precipitate UGTs from solubilized rat liver microsomes. The data obtained indicated that CYP3A2, CYP2B2, CYP2C11/13, and CYP1A2 all co- precipitated with UGTs. However, there were large differences in the levels of UGTs that co-immunoprecipitated with each isoform (Ishii et al., 2007). These overall findings support the hypothesis of UGT-CYP interactions facilitating multistep

metabolism. Interestingly, the interactions of DMEs may be one of the major contributing factors responsible for inter-individual differences in drug sensitivity that cannot be explained by genetic variation (Ishii et al., 2005).

UGT-UGT Interactions

Several studies have proposed that the UGTs themselves dimerize within the endoplasmic reticulum (ER). It was demonstrated at the beginning of the 1980s that rat liver UGTs tended to form aggregates and most likely existed as units larger than monomers (Matsui and Nagai, 1986; Matern et al., 1982; Ikushiro et al., 1997).

Radiation inactivation analysis of the UGTs, a method by which to determine the molecular masses of membrane-bound enzymes in situ, later revealed significant molecular-weight differences among enzymes that were mathematical multiples of each other, suggesting that the UGTs were composed of one to four subunits of similar molecular weights (Peters et al., 1984). Subsequent utilization of this method by

Gschaidmeier and Bock indicated that UGTs are functional as dimers in monoglucuronide formation and as tetramers in diglucuronide formation

(Gschaidmeier and Bock, 1994). Interactions between the UGT1s and UGT2B1 in rat microsomes were studied by immunopurification procedures with anti-peptide antibodies and chemical cross-linking experiments and revealed direct interactions between the UGT1A subfamily of enzymes and UGT2B1 (Ikushiro et al., 1997). In

1997, Meech and Mackenzie performed mutation, co-expression, and SDS-PAGE analyses to propose that UGTs formed catalytically active dimers via their amino- terminal domains (Meech and Mackenzie, 1997). Knowing that the highly variable,

substrate-binding N-terminus has been implicated in dimerization, it is not surprising that disrupting interactions within this region could alter Km values and substrate binding specificity (Bock and Kohle, 2009). In 2007, Operaña et al. utilized FRET and co-immunoprecipitation experiments to investigate intermolecular interactions between UGT1A proteins in COS cells. Homo-dimerization was observed between all the UGT1As. These studies also revealed the promiscuous nature of UGT1A1 to heterodimerize with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. Additionally, co-expression of UGT1A1 with UGT1A7 increased 2- napthol glucuronidation in comparison to both single expression systems, implicating the potential role of oligomerization on activity (Operaña et al., 2007). Studies by

Fujiwara et al. in 2010 of protein-protein interactions between human UGT2B7 and

UGT1As via double expression experiments in HEK293 cells revealed that UGT2B7 formed homo-oligomers, as well as hetero-oligomers, with UGT1A1, UGT1A4,

UGT1A6, and UGT1A9. Kinetic analysis revealed co-expression of UGT1A enzymes altered UGT2B7-catalyzed zidovudine O-glucuronidation. Co-expression of UGT2B7 also modified the kinetics of estradiol 3-O-glucuruonidation by UGT1A1, imipramine

N-glucuronidation by UGT1A4, serotonin O-glucuronidation by UGT1A6, and propofol O-glucuronidation by UGT1A9, clearly demonstrating the effect of interactions between human UGT2B7 and UGT1A on activity (Fujiwara et al.,

2010a). Overall, these findings support in vitro UGT-UGT interactions and their capacity to modulate activity. However, concrete evidence of interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated.

The ability to study UGT protein interactions in physiologically relevant, in vitro

systems will help to further address their functional relevance as well as aid in identifying potential disconnects between UGT enzymology in single enzyme versus whole cell systems.

RNA Interference as a Tool to Study Glucuronidation

Research over the last decade has led to exciting new discoveries on the role of double-stranded RNA (dsRNA) in the cell. The diverse effects of dsRNA on gene expression are now known to include orchestrating epigenetic changes, repressing translation, and directing mRNA degradation in a sequence-specific manner.

However, in the 1990s, injection of large amounts of antisense single-stranded RNA

(ssRNA) had been the traditional approach by which to study gene function in nematodes (Rao and Sockanathan, 2005). Gene silencing, termed RNA interference

(RNAi), was achieved through ssRNA injection, in the hope that it would pair with its complementary mRNA, block translation, and lead to an effective loss-of-function

(Nellen and Lichtenstein, 1993). Experiments by Guo and Kemphues to assess par-

1’s role in establishment of anterior-posterior polarity of Caenorhabditis elegans (C. elegans) embryo revealed that both sense and antisense RNA preparations were capable of causing interference (Guo and Kemphues, 1995). Additional work has also shown that, unlike cellular mRNAs, which tend to have relatively short half-lives, ssRNAs exhibit prolonged silencing effects and can even be inherited from one generation to the next (Seydoux and Fire, 1994). Despite the fact that the intrinsic differences between endogenous RNA and the interference-inducing substrate remained largely unexplained, antisense-mediated silencing still continued to be a

widely used technique. Subsequently, in 1998 and at the time of the completion of the

C. elegans genome project, Fire and Mello made an innovative discovery. In an attempt to address the observed discrepancies in previous silencing studies, they unfolded a new technology based on the silencing of specific genes by dsRNA. Fire et al. had previously noted that ssRNA samples prepared with bacteriophage RNA polymerases were often contaminated with ectopic transcripts. They hypothesized that the presence of dsRNAs could be the reason why both sense and antisense RNAs were capable of inducing silencing. To test their hypothesis, C. elegans were injected with either single- or double-stranded RNA targeting the unc-22 gene. Over a 100-fold greater silencing effect was seen with dsRNA than with either strand individually.

Furthermore, it was observed that the silencing effect could cross into the adult’s gonads and be transferred to the worm’s progeny after initial injection in its head, thus implicating an active transport mechanism necessary for achieving long-distance effects (Rao and Sockanathan, 2005). These results fueled further research, as RNAi became the standard means by which to investigate gene function in C. elegans. In

1999, studies of the interference process in C. elegans mutants resistant to dsRNA- mediate interference helped identify the genes required in RNAi (Tabara et al., 1999).

Earlier reports had observed gene silencing mediated by unknown substrates in several diverse organisms, including insects, plants, and fungi, but sequence comparison with the recently identified C. elegans’ dsRNA-mediated silencing genes revealed similar machinery, confirming dsRNA-mediated silencing in other organisms (Hamilton and

Baulcombe, 1999; Fagard et al., 2000). RNAi has since been discovered in a wide variety of species, including fruit flies (Kennerdell and Carthew, 1998; Misquitta and

Paterson, 1999), trypanosomes (Ngo et al., 1998), planaria (Sanchez Alvarado and

Newmark, 1999), hydra (Lohmann et al., 1999), zebrafish (Wargelius et al., 1999), and mice (Wianny and Zernicka-Goetz, 2000) and appears to be related to the gene silencing phenomena observed in plants (Vaucheret et al., 1998; Waterhouse et al.,

1998; Waterhouse et al., 1999; Baulcombe, 1999) and fungus (Cogoni et al., 1996;

Cogoni and Macino, 1999a; Cogoni and Macino, 1999b; Zamore et al., 2000). Early application of dsRNAs of varying sizes (38-1,662 bp) to commonly used mammalian cell culture systems, including HEK293, NIH/3T3 (mouse fibroblast), BHK-21

(Syrian baby hamster kidney), and CHO-K1 (Chinese hamster ovary) cells had surprisingly failed to result in potent and specific interference. This apparent lack of

RNAi in mammalian cell culture was unexpected, as RNAi had been previously observed in mouse oocytes and early embryos (Wianny and Zernicka-Goetz, 2000;

Svoboda et al., 2000). However, researchers found that dsRNAs greater than 30 bp in the cytoplasm of mammalian cells provoked a potent interferon response (Stark et al.,

1998; Reynolds et al., 2006). Today, it is well established that RNAi not only controls mRNA levels, but also serves as a protective mechanism against viral infections. To address the issues occurring with siRNA mammalian transfection, Elbashir et al. expanded on their previous experiments in which they had achieved successful RNAi with smaller, 21- and 22-bp RNA fragments in a Drosophila in vitro system (Elbashir et al., 2001b). They demonstrated that transfection with 21-bp siRNA duplexes specifically suppressed expression of endogenous and heterologous genes in different mammalian cell lines, including HEK293 and HeLa cells (Elbashir et al., 2001a).

Therefore, 2001 marked the first description of siRNA use in mammalian cell culture

and a surge of siRNA use in mammalian cells soon followed. Ultimately, these exciting discoveries implicate dsRNA-mediated silencing as more than just a new tool for loss-of-function studies, but rather an ancient phenomenon with an important, protective biological role (Rao and Sockanathan, 2005).

siRNA technology today provides a novel tool for systematically deciphering the functions and interactions of thousands of genes (McManus and Sharp, 2002).

More modern uses of siRNA include targeting the expression of over expressed molecular targets in cancer therapy or for discerning the importance of an enzymatic pathway in an in vitro pharmacology assay (Rao et al., 2009). However, to date, the use of siRNA to examine DME activity is fairly limited. Gene silencing via siRNA down regulation to study glucuronidation has been previously reported in both HeLa cells as well as in a Caco-2 cell system (Liu et al., 2007; Jiang et al., 2012).

Identification of UGT1A6 as the primary UGT isoform involved in the glucuronidation of flavanoids in Caco-2 cells was determined through the use of

RNAi, after a significant decrease in apigenin glucuronidation was observed with down regulation of UGT1A6 expression in tissues culture (Liu et al., 2007). In the absence of selective UGT inhibitors, the use of siRNA technology provides a tool to selectively silence individual UGT isoforms that would allow for the assessment of changes in the enzyme activity both of the targeted UGT as well as other UGTs interacting on a protein level with the silenced UGT.

The Xenobiotic Receptors

Most animals, including humans, are exposed daily to a multitude of chemicals

in the air, water, or food. While some of these chemicals are signaling molecules that carry valuable information about the animal’s environment, such as the presence of food, predators, or members of the opposite sex, others are toxic and must be avoided or eliminated. Mammalian enzymatic defenses have evolved to facilitate the biotransformation and elimination of toxic compounds encountered in the environment

(Hahn, 2002). Receptors that function as sensors of toxic byproducts derived from both endogenous and exogenous metabolism have been termed xenobiotic receptors.

These include but are not limited to the farnesoid X receptor (FXR), liver X receptor

(LXR), peroxisome proliferator activation receptors (PPARs), constitutive androstane/active receptor (CAR), pregnane X receptor (PXR), nuclear factor- erythroid 2-related factor 2 (Nrf2), and aryl hydrocarbon receptor (AhR) (Tolson and

Wang, 2010). Among these, AhR, CAR, PXR and Nrf2 are the most extensively studied for their roles in the induction of cytoprotective genes, including the DMEs

(Shen and Kong, 2009).

In order to understand how the DMEs are regulated, it is necessary to address the signaling mechanisms involving the xenobiotic receptors at the molecular level

(Xu et al., 2005). The xenobiotic receptors comprise a gene superfamily encoding for ligand-activated transcription factors, which transform endogenous and exogenous stimuli into cellular responses by regulating the expression of their target genes

(Levine and Perdew, 2001; Wang and LeCluyse, 2003). Transcriptional regulation of gene expression by these receptors plays an essential role in the metabolism and clearance of many drugs and xenobiotics that are introduced into the body, for the purpose of protecting the body from the environmental insults (Li et al., 1998;

Rushmore and Kong, 2002; Wang and LeCluyse, 2003).

The Aryl Hydrocarbon Receptor

The adaptive function of the aryl hydrocarbon receptor (AhR) has been studied for more than 30 years. Research initially sparked by the observed extraordinary toxic potency of chlorinated dibenzo-p-dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD) in animals (Higginbotham et al., 1968; Schwetz et al., 1973). Later focus on the potency of TCDD for eliciting biochemical effects (Poland and Glover, 1973a;

Poland and Glover, 1974), such as induction of aryl hydrocarbon hydroxylase activity that is now known to be catalyzed primarily by CYP1A1 (Poland and Glover, 1973b;

Poland and Glover, 1977; Goldstein et al., 1977) paved the way for the discovery of

AhR. The existence of AhR was first identified when PAH responsive strains of mice were found to express a receptor that bound TCDD with greater affinity when compared to PAH nonresponsive mice (Poland et al., 1974). Poland et al. firmly established the presence of AhR via in vitro experiments on binding of [3H]TCDD to hepatic cytosols from PAH responsive mice, which revealed a small pool of high- affinity binding sites that stereospecifically and reversibly bound TCDD (Poland et al.,

1976). Since the 1976 breakthrough, the mechanism by which AhR regulates the induction of adaptive enzymes has been under investigation (Whitlock, 1999).

In 1986, AhR was successfully purified upon the development of a photoaffinity ligand used to monitor the receptor (Poland et al., 1986), thus enabling the subsequent cloning and characterization of the receptor (Ema et al., 1992; Burbach et al., 1992). AhR amino acid sequence analysis revealed a region similar to the basic

region/helix-loop-helix (BR/HLH) motif found in many transcription factors that dimerize for function as well as that the N-terminal domain of AhR exhibited extensive sequence similarity to the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein and two regulatory proteins of Drosophila, Sim and Per (Schmidt and Bradfield, 1996; Hankinson, 1995). In addition, it was determined that AhR binds an agonist at a domain that lies within this conserved N-terminal domain. These findings confirmed that AhR functions as a ligand-activated transcription factor with a helix-loop-helix motif similar to those found in a variety of DNA-binding proteins

(Burbach et al., 1992; Ema et al., 1992). The AhR N-terminal region contains the basic residues that contribute to DNA binding, while the HLH domain facilitates protein-protein dimerization with other proteins and transcription factors (Murre et al.,

1989a; Murre et al., 1989b; Davis et al., 1990). The N-terminal region also contains the AhR nuclear localization signal and a nuclear export signal sequence (Ikuta et al.,

1998). The C-terminal portion contains the Per-Arnt-Sim (PAS) region and influences protein-protein interactions, DNA recognition, and ligand binding (Burbach et al.,

1992; Denis et al., 1988; Perdew, 1988). It is now known that AhR is a member of the basic-helix-loop-helix (bHLH)-PAS gene superfamily of transcription factors (Gu et al., 2000; Crews, 1998; Hahn, 1998). Proteins with PAS-related domains occur in organisms as diverse as animals, plants, fungi, bacteria, and archea, implicating the importance of adaptive inducibility in defense (Crews, 1998; Somers et al., 2000;

Ballario et al., 1998; Pellequer et al., 1998; Crosthwaite et al., 1997; Taylor and

Zhulin, 1999). It was originally believed that AhR had relatively narrow structural specificity (Poland and Knutson, 1982); however, it is now known that the receptor

recognizes an impressive range of chemical structures, including non-aromatic and non-halogenated compounds (Denison and Nagy, 2003). In the context of the adaptive function of AhR, such substrate promiscuity is understandable and emphasizes its role as an environmental sensor.

The mechanism of AhR signaling has been extensively studied with respect to

CYP1A1 induction. Immunofluorescence microscopy using AhR-specific antibodies has revealed that the unbound receptor is localized in the cytosol (Pollenz et al., 1994).

Unbound AhR is retained in the cytosol through interactions with three heat shock proteins: Hsp90, which maintains AhR in a high affinity ligand binding state and prevents nuclear translocation and dimerization with ARNT, co-chaperone protein p23, and XAP2, an immunophilin-related protein involved in the regulation of AhR turnover (Perdew, 1988; Meyer et al., 1998, Meyer and Perdew, 1999; Heid et al.,

2000; Dull et al., 2002). Ligand binding induces a conformational change of the receptor that results in increased DNA binding affinity and decreased rate of ligand dissociation (Davis et al., 1990), resulting in nuclear translocation of ligand-bound

AhR and subsequent heterodimerization with its partner ARNT (Whitlock, 1999;

Gonzalez et al., 1996; Schmidt and Bradfield, 1996; Hankinson, 1995; Okey et al.,

1994). ARNT was originally thought to be necessary for nuclear translocation of AhR

(Hoffman et al., 1991), but it is now known that it is not required (Reyes et al., 1992).

The AhR/ARNT heterodimer complex recognizes and binds to distinct DNA sequences called xenobiotic response elements (XREs), leading to the transactivation and induction of target genes containing XREs in their promoter region.

Electrophoretic mobility shift assays demonstrated that activated AhR recognized a

specific DNA motif containing the XRE sequence: 5’-TGCGTG-3’ (Denison et al.,

1989). Utilization of short segments of upstream regions of mouse and rat CYP1A1 genes fused to the chloramphenicol acetyl transferase reporter gene that became activated upon TCDD or PAH treatment allowed for the initial identification of these

XRE consensus sequences in both the mouse and human CYP1A1 gene (Fujisawa-

Sehara et al., 1987; Denison et al., 1988).

The XRE motif has since been identified in the regulatory regions of the

UGTs, having first been observed in rat UGT1A6 (Emi et al., 1996), human UGT1A6

(Munzel et al., 1998; Munzel et al., 2003), and human UGT1A1 (Yueh et al., 2003).

Treatment with the flavonoid, chrysin has been found to induce UGT1A1 in both

HepG2 and Caco-2 cells (Walle et al., 2000; Galijatovic et al., 2001; Galijatovic et al.,

2000). Since flavonoids are also capable of inducing CYP1A1 (Allen et al., 2001) in a

CYP1A1-luciferase reporter HepG2 cell line (Chen and Tukey, 1996), it was hypothesized that induction of UGT1A1 may occur through a similar mechanism. In

2003, Yueh et al. demonstrated induction of UGT1A1 in HepG2 cells treated with specific AhR ligands, such as TCDD, β-naphthoflavone (BNF), and B[a]P metabolites, by monitoring increases in UGT1A1 mRNA, protein, and catalytic activity. Yueh and coworkers also performed nucleotide sequence analysis of the

UGT1A1 enhancer region, which revealed the presence of an XRE within that region.

Mutation of this sequence eliminated binding of AhR and subsequent generation of enhancer constructs containing the same mutation resulted in a loss of TCDD and

BNF induction of reporter gene activity (Yueh et al., 2003). To date, human UGT1A1,

UGT1A3, UGT1A4, UGT1A6, and UGT1A9 have been shown to be regulated by AhR

(Mackenzie et al., 2010; Yueh et al., 2003; Bonzo et al., 2007; Ou et al., 2010; Chen et al., 2005; Sugatani et al., 2004; Lankisch et al., 2008), thus implicating the importance of UGT-AhR interactions in protection against environmental insults.

Nuclear Receptors

The nuclear receptor superfamily is one largest group of transcription factors, with 49 distinct members presently identified in the human genome (Maglich et al.,

2001; Robinson-Rechavi et al., 2001), and is responsible for regulating development and metabolism through control of gene expression. Many of these proteins directly bind to signaling molecules, which, because of their small, lipophilic character, can then easily enter target cells. Therefore, unlike membrane-bound receptors, the nuclear receptors are intracellular and function to directly control the activity of target genes (Mangelsdorf and Evans, 1995). While modern molecular biology studies of these receptors began around 15 years ago with the cloning of the estrogen and glucocorticoid receptors (Mangelsdorf et al., 1995), the endogenous ligands for these receptors have been studied for more than 60 years, since hormone-regulated pathways were the target of interest for medicinal chemists even before the receptors were identified (Evans, 1988). Receptors characterized prior to the identification of their ligands were referred to as orphan receptors, and as their natural and synthetic ligands have become known, many of them have now been adopted (Chawla et al.,

2001). Over time, the synthesis and use of new ligands have facilitated in the discovery of new roles and relationships for the nuclear-receptor superfamily,

ultimately emphasizing their importance as master regulators of genes (Weatherman et al., 1999).

The nuclear receptors primarily act through the direct association with specific

DNA sequences known as hormone response elements (HREs) (Evans, 1988; Beato,

1991). The combined achievements of the discovery of puff induction by ecdysone in giant chromosomes of insects (Clever and Karlson, 1960), the characterization and purification of hormone receptors, and the cloning of hormonally regulated genes culminated in the identification of hormone responsive sequences in the vicinity of genes regulated by steroid hormones (Beato, 1989). Since then, the speed of progress in the field has been extraordinarily fast, with dozens of regulatory elements for steroid hormones having been described and the cDNAs for virtually all known hormone receptors having been cloned (Evans, 1988). The powerful techniques of genetic engineering in combination with these clones provided insight into the molecular and functional architecture of the nuclear hormone receptors. In 1978, several protein chemical studies had already suggested that the steroid hormone receptors were structurally organized into different domains (Wrange and Gustafsson,

1978; Carlstedt-Duke et al., 1982; Wrange et al., 1984; Carlstedt-Duke et al., 1987).

This prediction was confirmed in 1986 and 1987, with the comparison of amino acid sequences of various hormone receptors (Kumar et al., 1986; Hollenberg et al., 1987;

Rusconi and Yamamoto, 1987). All analyzed nuclear receptors to date have been found to be structured in a similar way, exhibiting a variable N-terminal region, a short and well-conserved cysteine-rich central domain, and a relatively well-conserved

C-terminal half (Evans and Hollenberg, 1988), but can essentially be described in

terms of their DNA-binding domain (DBD) and a ligand-binding domain (LBD). The

DBD targets the receptor to specific HREs and contains several conserved cysteine residues. Eight of these cysteines are organized into two zinc fingers (Berg, 1989), a structural motif commonly identified in other genes for regulatory proteins, as well as enzymes (Klug and Schwabe, 1995). Identification of these repetitive zinc-binding domains within the receptor’s central domain (Miller et al., 1985), in addition to in vitro binding studies and functional evidence, verified that the domain was responsible for receptor DNA binding (Evans and Hollenberg, 1988). The C-terminal half of the receptor encompasses the LBD that possesses the essential property of hormone recognition and ensures both specificity and selectivity of the physiologic response. In its simplest terms, the LBD can be thought of as a molecular switch that when, bound by a ligand, shifts the receptor to a transcriptionally active state (Mangelsdorf et al.,

1995). Photo-crosslinking experiments (Carlstedt-Duke et al., 1988; Simons et al.,

1987) as well as mapping of the LBD have helped to identify those residues responsible for direct ligand binding and those for facilitating interactions. Studies performed by Fawell et al. found that single amino acid substitutions of residues in the

N-terminal half of the estrogen receptor, but not the C-terminal half, prevented dimerization (Fawell et al., 1990). Ultimately, the sequence-specific DNA binding properties of the nuclear receptors are determined by these two conserved domains

(DBD and LBD), which function in an interdependent manner to mediate protein-

DNA and protein-protein interactions. The minimal target sequence recognized by the nuclear receptor DBD consists of a six base pair sequence. Several features of the core recognition motif are conserved for all members of the nuclear receptor

superfamily. The detailed characterization of response elements within target genes that mediate their transcriptional activation has led to the identification of three distinct DNA-binding modes that are distinguished by whether the interactions occurring between response elements and nuclear receptors are monomers, homodimers, or heterodimers (Glass, 1994), the last two being the most common associations. Homodimeric recognition of response element sequences is typified by steroid hormone receptors and was first demonstrated in the cases of the estrogen receptor and glucocorticoid receptor (Kumar and Chambon, 1988; Tsai et al., 1988).

Steroid HREs are generally comprised of a pseudo-palindromic arrangement of two core recognition sequences. Crystallographic analysis revealed that each DBD of the dimer makes similar contacts with one of the core recognition motifs, forming in a symmetric structure (Luisi et al., 1991; Schwabe et al., 1993). Heterodimeric interactions between nuclear receptors and DNA response elements are classically demonstrated by the retinoic acid receptor (RAR), thyroid hormone receptor (TR), vitamin D receptor (VDR), and several of the orphan nuclear receptors, including the pregnane X receptor (PXR) and constitutive androstane receptor (CAR). These receptors form heterodimers with the retinoid X receptors (RXR) and bind to response elements consisting of direct, everted, or inverted repeat arrangements of the core consensus sequence (Forman and Evans, 1995; Mackenzie et al., 2010; Yu et al.,

1991; Kliewer et al., 1992; Leid et al., 1992; Zhang et al., 1992; Marks et al., 1992).

The identification of these distinct DNA-binding modes subsequently revealed three key response element features that regulate the specificity of DNA recognition by a particular set of nuclear receptors: the precise sequence of the core recognition motif,

the orientation of core recognition motifs with respect to each other, and the spacing between those motifs (Glass, 1994). For example, synthetic DNA sites consisting of direct repeats of consensus hexamers separated by 3, 4, or 5 base pairs would be referred to as DR-3, DR-4, and DR-5. These distinct arrangements dictate the preferential responses to specific receptors (Baes et al., 1994).

The Pregnane X Receptor and the Constitutive Androstane Receptor

The pregnane X receptor (PXR, NR1I2) belongs to the orphan nuclear receptor superfamily of ligand-activated transcription factors (Kliewer et al., 1998) and has since been shown to play an essential role in both endobiotic (Zollner et al., 2006; Iyer et al., 2006; Cho et al., 2009; Bhalla et al., 2004; Bachmann et al., 2004; Xie et al.,

2001; Sugatani et al., 2005; Matic et al., 2007) and xenobiotic (Francis et al., 2003;

Jones et al., 2000; Tolson and Wang, 2010; Chirulli et al., 2005) metabolism in humans, mice, and rats. Mouse PXR (mPXR) was first discovered and cloned in 1997 based on sequence homology with other nuclear receptors and was found to be activated by a variety of compounds, including natural and synthetic glucocorticoids, steroids, pregnane derivatives, anti-glucocorticoids, macrocyclic antibiotics, antifungals, and herbal extracts (Kliewer et al., 1998; Jones et al., 2000; Kliewer et al.,

1999; Lehmann et al., 1998; Bertilsson et al., 1998; Blumberg et al., 1998; Moore et al., 2002). The human PXR (hPXR) ortholog was subsequently reported as the steroid and xenobiotic receptor (SXR) and pregnane activated receptor (PAR), which both exhibited structural features and activation patterns similar to mPXR (Bertilsson et al.,

1998; Blumberg et al., 1998). Xie and colleagues finally confirmed SXR/PAR to be

orthologous to mPXR with PXR knockout and transgenic mouse models. Targeted disruption of the mPXR abolished CYP3A induction by prototypic inducers, such as dexamethasone or pregnenolone-16α-carbonitrile (PCN), while an activated form of

SXR caused constitutive upregulation of CYP3A gene expression and enhanced protection against toxic xenobiotic compounds in transgenic mice (Xie et al., 2000a).

PXR has since been cloned from a wide array of species, including rabbit, birds, amphibians, fish, pig, rhesus monkey, and dog (Jones et al., 2000; Savas et al., 2000;

Handschin et al., 2000; Moore et al., 2002; Willson et al., 2001). Ligand-activated

PXR translocates from the cytoplasm to the nucleus of the cells (Squires et al., 2004), where it then binds to DNA response elements as a heterodimer with RXR.

PXR was initially thought of as “conventional” nuclear receptor, as it appeared to exert its effects through a similar mechanism of action as the other steroid hormone receptors. However, cross-species comparisons have revealed surprising differences in the amino-acid sequence of their ligand-binding domains, indicating a relatively rapid and divergent evolution of these proteins (Willson and Kliewer, 2002). The ever-evolving library of structurally diverse PXR ligands has come to distinguish it as a unique, promiscuous, but integral mediator of inductive expression of many DMEs and transporters (Tolson and Wang, 2010). Microarray gene profiling analysis on liver samples derived from mice expressing a constitutively active variant, VP-hPXR, identified approximately 150 gene tags that were expressed in a PXR-dependent manner, including a spectrum of biologically important Phase I and II DMEs

(Rosenfeld et al., 2003). The extremely variable nature of PXR in ligand and target gene recognition verifies its ability to serve as a xenobiotic sensor (Tolson and Wang,

2010).

In the nuclear receptor superfamily tree, CAR (NR1I3) is the closest relative to

PXR. Initially named MB67 in 1994, it was isolated by screening a cDNA library with a nuclear receptor DBD-based oligonucleotide as a probe (Baes et al., 1994).

This receptor was later re-designated as constitutive activated receptor (CAR), based on its ability to form a heterodimer with RXR that bound retinoic acid response elements to transactivate target genes in the absence of ligand in transfection assays

(Baes et al., 1994; Choi et al., 1997). The first class of CAR ligands was then discovered in 1998, which included androstanol and androstenol. Interestingly, these compounds were characterized as inverse agonists because androstanol and androstenol were capable of inhibiting the constitutive activity of CAR by promoting co-activator release from the LBD (Forman et al., 1998). Appropriately, this receptor is also referred to as the constitutive androstane receptor. Major progress in understanding the physiological roles of CAR came with the observation that CAR activation was linked to CYP2B gene induction by PB and PB-like inducers

(Honkakoski et al., 1998). Purification of CAR from hepatocytes by Negishi and colleagues identified it as a factor that is associated with the PB-responsive CYP2B regulatory element (Zelko and Negishi, 2000; Sueyoshi and Negishi, 2001), and it was subsequently shown to bind to the promoter as a heterodimer with RXR (Honkakoski et al., 1998). Interestingly, endogenous CAR was found to reside in the cytoplasm of hepatocytes and therefore unable to affect gene transcription. Only upon exposure to

PB did CAR translocate from the cytoplasm to the nucleus via a phosphorylation- dependent mechanism (Kawamoto et al., 1999; Zelko et al., 2001). Although there is

no evidence that PB directly binds CAR, the PB-induced translocation of the receptor results in increased CYP2B gene transcription in the cell nucleus. Reconstitution of this phenomenon with cell-based reporter assays has proved difficult, as CAR expressed by cells that are either stably or transiently transfected with a CAR expression vector resides in the nucleus, regardless of its activation state (Sueyoshi et al., 1999; Tzameli et al., 2000). The generation of knockout mice has thus provided definitive evidence of the role of CAR in regulating CYP2B expression in vivo, as Car

-/- mice exhibited no Cyp2b induction with PB or PB-like inducer TCPOBOP (1,4- bis(2-(3,5-dichloropyridyloxy))benzene) (Wei et al., 2000). These findings triggered a wealth of subsequent studies that explored the role of CAR on xenobiotic metabolism

(Honkakoski et al., 2003; Qatanani and Moore, 2005). In addition to both CAR and

PXR being highly expressed in the liver and small intestine, which are two key tissues greatly involved in drug metabolism (Lehmann et al., 1998), these two receptors share significant cross-talk in both target gene recognition by binding to similar responsive elements in their target gene promoters and in accommodating a diverse array of xenobiotic activators (Xie et al., 2000b; Wang and LeCluyse, 2003), further implicating their importance in xenobiotic defense mechanisms.

UGT1A1 is one of the most extensively characterized UGT isoforms, due to its important physiological role in the clearance of bilirubin as well as xenobiotics

(Mackenzie et al., 2003; Radominska-Pandya et al., 2005). While PB treatment has been extensively used over the last 40 years to treat hyperbilirubinemia resulting from

UGT1A1 deficiencies (Yaffe et al., 1966), the mechanism by which PB induces

UGT1A1 activity has only been realized within the last decade. Sugatani et al.

(Sugatani et al., 2001) first reported that PB-mediated induction of UGT1A1 could be attributed to a 290-bp enhancer sequence upstream of the UGT1A1 promoter. This sequence contained three putative nuclear receptor-binding motifs and was also responsive to CAR activation. In 2003, Xie and colleagues demonstrated that

UGT1A1 expression was induced at both the mRNA and protein levels, in both transgenic VP-hPXR mice and in rifampicin-treated humanized PXR mice (Xie et al.,

2003). It was indepdently demonstrated that rodent-specific PXR agonist PCN increased UGT activity and expression only in wild-type, but not in PXR null mice

(Chen et al., 2003). In addition to UGT1A1, UGT1A9 expression has been observed to increase with PXR activation (Chen et al., 2003). UGT1A3, UGT1A4, UGT1A6 and

UGT1A9 may also be PXR target genes, although exact DNA responsive elements required for these effects have yet to be found (Shelby and Klaassen, 2006; Vyhlidal et al., 2004; Buckley and Klaassen, 2009).

UGT1A1 was the first UGT enzyme that was defined as a CAR target gene through the receptor’s ability to recognize and bind to a distal phenobarbital- responsive enhancer module of UGT1A1 (Sugatani et al., 2001). Reduction in

UGT1A1 expression is associated with various clinical conditions, including Gilberts' syndrome characterized by mild, unconjugated hyperbilirubinemia. Polymorphism analysis of the UGT1A1 promoter revealed a SNP of −3263 T > G located within the

CAR enhancer sequence whose frequency was significantly higher in patients with

Gilbert's syndrome (58%) in comparison with healthy volunteers (17%) (Sugatani et al., 2002). In addition, this mutation significantly reduced CAR-mediated transcriptional activation of UGT1A1 in cell-based luciferase assays, indicating that

interplay occurs between gene polymorphism and nuclear receptor-mediated induction of UGT1A1. Following these findings, Qatanai and colleagues showed that CAR activation increased the major pathway of bilirubin clearance by inducing the expression of UGT1A1, MRP2, SLC21A6, GSTA1, and GSTA2 (Qatanani et al., 2005).

Moreover, Car -/- mice pretreated with PB or TCPOBOP did not exhibit the marked increase in bilirubin clearance seen in wild-type mice. Bilirubin itself has also been documented as a CAR activator, suggesting a potential protective feedback mechanism when bilirubin accumulates in the body (Huang et al., 2003). The list of

UGTs as potential CAR target genes has recently been expanded to include UGT1A3,

UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A10, UGT2B1, and UGT2B5 (Shelby and Klaassen, 2006; Qatanani et al., 2005). Overall, the role of PXR and CAR as mediators in the induction of UGTs expands the scope of these xenosensors. Mice lacking either PXR or CAR were hypersensitive to treatment with various xenobiotics, including the anesthetic tribromoethanol and the muscle relaxant zoxazolamine (Wei et al., 2000; Xie et al., 2000a), ultimately underscoring the importance of these two nuclear receptors in defending the body against a broad array of potentially harmful xenobiotics (Maglich et al., 2002; Tolson and Wang, 2010).

The Oxidative Stress Sensor, NF-E2 related factor-2

NF-E2 related factor-2 (Nrf2) is a 66-kDa ubiquitous protein belonging to the small family of basic leucine zipper (bZip) transcription factors that binds an AP1/NF-

E2 tandem repeat sequence (Gourdon et al., 1992; Moi and Kan, 1990; Ikuta and Kan,

1991) and was originally implicated in regulating globin gene expression in

hematopoietic cells (Andrews et al., 1993; Moi and Kan, 1990; Ney et al., 1990;

Collis et al., 1990; Chang et al., 1992). Studies with both transfected cells and transgenic mice found a novel protein that was capable of activating this enhancer sequence within the locus control region of the globin gene, called nuclear factor- erythroid 2 (NF-E2) (Holtzclaw et al., 2004). The tandem repeat NF-E2 recognition sequence was then used to screen a lambda gt11 cDNA expression library from K562 cells, which resulted in the identification of several other DNA binding proteins, one of which was called NF-E2-related factor 2 (Nrf2) (Moi et al., 1994). While NF-E2 expression was restricted to erythroid and megakaryocytic cells, Nrf2 was ubiquitously expressed in a wide range of tissues, many of which are sites of expression for Phase II detoxification genes (Chan et al., 1996; Chan and Kan, 1999;

McMahon et al., 2001; Moi et al., 1994; Chan et al., 1993). In addition, Nrf2 -/- mice generated by Chan et al. were not anemic, developed normally, and reproduced, indicating that Nrf2 was not required for the production of red blood cells (Chan et al.,

1996). However, additional challenge experiments in Nrf2 null mice did elucidate its importance in the oxidative stress response. In 1999, Chan and Kan observed that

Nrf2 -/- mice died of acute respiratory distress syndrome when administered doses of the antioxidant butylated hydroxytoluene (BHT) that were normally tolerated by wild- type mice (Chan and Kan, 1999). Subsequent gene expression studies also showed that the expression of many detoxification enzymes becomes altered in the Nrf2 null model. Several in vivo works have since shown that Nrf2 deficiency results in altered

DME gene expression and subsequent detrimental conditions due to the inability to properly respond to stress. Mice deficient in Nrf2 have significantly lower and

generally un-inducible levels of Phase II enzymes (McMahon et al., 2001; Chanas et al., 2002) and are much more sensitive than their wild-type counterparts to oxidative stress (Ishii et al., 2000; Hirayama et al., 2003), pulmonary toxicity (Cho et al., 2002), and hepatotoxicity (Chan and Kan, 1999; Enomoto et al., 2001). Nrf2 knockout mice were more susceptible to stomach tumor development in response to B[a]P administration due to a lack of protection against tumor formation, as protection was seen to result from induction of Phase II and antioxidant enzymes by oltipraz or sulforaphane (Ramos-Gomez et al., 2001; Fahey et al., 2002). The use of Nrf2 deficient mice highlights the crucial importance of elevated Phase II gene expression in cytoprotection.

The Nrf2 AP1-NFE2 recognition motif was later found to be a subset of the antioxidant response element (ARE) (Rushmore et al., 1991; Xie et al., 1995).

Promoter analysis of genes encoding for antioxidant enzymes led to the identification of a DNA element that regulated their basal expression and coordinated induction in response to antioxidants and xenobiotics (Dhakshinamoorthy et al., 2000; Jaiswal,

2000). Initially thought to be a unique XRE, this enhancer module was first identified in the promoters of genes encoding the two major detoxication enzymes, GSTA2 and

NADPH:quinone oxidoreductase 1 (NQO-1) (Rushmore et al., 1990; Friling et al.,

1990; Favreau and Pickett, 1993; Li and Jaiswal, 1992). These findings suggested that the regulation of gene expression by planar aromatic compounds, such as BNF and

MC, was mediated by this unique DNA sequence that was distinct from the classical

XRE sequence (Favreau and Pickett, 1991). Later work by Rushmore and Pickett found the sequence to also be responsive to hydrogen peroxide and phenolic

antioxidants, such as t-butylhydroquinone (tBHQ), thus leading to its officially designation as the ARE (Rushmore and Pickett, 1990).

Elucidating the mechanism controlling Nrf2 activity was central to understanding how terrestrial organisms sensed destructive oxidative stress and subsequently activated an intrinsic cellular defense (Itoh et al., 1999). Itoh et al. had observed that upon exposure to electrophilic agents, DNA-binding activity of Nrf2 was significantly induced, while Nrf2 steady-state mRNA levels remained constant.

These results indicated that signals from oxidative stress agents might be transduced through an unidentified cellular receptor to the Nrf2 protein, which in turn mediates the protective response. In order to explore the molecular mechanisms that activate

Nrf2 and thereby transduce oxidative stress signals, Itoh et al. started by examining the function of Nrf2’s six domains (Neh1–Neh6) via transient co-transfection experiments in which each region was fused to the DBD of the yeast transcription factor Gal4 or a luciferase reporter gene (Itoh et al., 1999). Neh4 and Neh5 were found to be independent activation domains, whereas the amino terminal Neh2 region was not. To determine Neh2’s function, the authors prepared an Nrf2 mutant from which the Neh2 domain was deleted. Wild-type and mutant Nrf2 cDNAs were independently co-transfected into QT6 quail fibroblasts or HD3 chicken proerythroblasts with the luciferase reporter construct. The cells transfected with the mutant Nrf2 exhibited much higher luciferase activity than those transfected with the wild-type DNA, suggesting that a cellular repressor that interacts with the Neh2 domain of Nrf2 may exist. The Gal4–Neh2 construct was subsequently used as bait in a yeast two-hybrid system to isolate the repressor protein. Of the 300 clones

recovered, 80 were analyzed and most contained the sequence of a single protein that was named Keap1 (Kelch-like ECH associated protein 1) because of its sequence homology with the drosophila actin-associated protein Kelch. Transfection of the

Keap1-GFP construct into QT6 fibroblasts resulted in Keap1 localization within cytosol, while transfection of a Neh2-GFP into 293T cells revealed uniform fluorescence throughout the cells. However, co-transfection of Keap1 with Neh2-GFP displayed almost exclusive cytoplasmic fluorescence. Following these observations, treatment with the electrophilic agent diethyl maleate (DEM) was found to release the

Nrf2 protein from the cytoplasm to the nucleus. Anti-Nrf2 immunoreactivity was detected predominately in the nucleus when Nrf2 expression plasmid alone was transfected into 293T cells, but when Keap1 was co-transfected with Nrf2, the cellular localization of Nrf2 was principally in the cytoplasm. Upon addition of DEM to the culture medium, Nrf2 was detected in the nucleus, even in the presence of co- transfected Keap1, thus showing DEM enables Nrf2 nuclear translocation from the cytoplasm (Itoh et al., 1999). Complementary to these findings, Wakabayashi et al. subsequently developed a Keap1-deficient mouse and showed that the absence of

Keap1 resulted in the constitutive accumulation of Nrf2 in the nucleus and therefore high expression levels of cytoprotective genes (Wakabayashi et al., 2003).

Additionally, examination of Keap1’s sequence revealed it to be extremely cysteine rich and thus lead to the conclusion that Keap1 had the potential to serve as a perfect sensor for inducers (Dinkova-Kostova et al., 2005). At its simplest, Keap1 retains the

Nrf2 transcription factor in the cytoplasm until increases in cellular oxidative stress cause Keap1 to dissociate, allowing for Nrf2 nuclear translocation. Nrf2 then binds to

the ARE present in the promoters of genes encoding several dozen cytoprotective proteins that enhance cell survival (Nioi et al., 2003; Kensler et al., 2007; Ishii et al.,

2000). Ultimately, these findings implicated the Keap1-Nrf2 complex as a cytoplasmic sensor system for oxidative stress.

Since Nrf2 was not only activated in response to H2O2, but specifically by chemical compounds with the capacity to either undergo redox cycling or be metabolically transformed to a reactive or electrophilic intermediate, this led researchers to believe that alterations in the cellular redox status due to elevated levels of reactive oxygen species (ROS) and electrophilic species and/or a reduced antioxidant capacity (e.g., glutathione) might be an important signal for triggering the transcriptional response mediated by this enhancer (Nguyen et al., 2000). The involvement of Nrf2 was further supported by Nrf2 transactivation of reporter genes linked to the ARE sequence (Venugopal and Jaiswal, 1996; Venugopal and Jaiswal,

1998), in vivo studies in which expression of several ARE-dependent genes was found to be severely impaired in Nrf2 -/- mice (Itoh et al., 1997; McMahon et al., 2001), and by chromatin immunoprecipitation assays demonstrating direct interaction between endogenous Nrf2 and the ARE in H4IIE cells (Nguyen et al., 2005).

Previous studies have shown that regulation of UGT expression is targeted by a number of xenobiotic receptors in response to xenobiotics, carcinogens, and stress signals. Induction of proteins following exposure to electrophiles and oxidants has been termed the antioxidant response and has been linked to Nrf2 activation

(Holtzclaw et al., 2004), as Nrf2 has been identified as the major regulator of cytoprotective genes encoding detoxification and antioxidant enzymes (Itoh et al.,

1995; Ishii et al., 2000). The induction of UGT activity in mice treated with natural or synthetic chemopreventive agents represented an initial indication that UGT activity may be among the cytoprotective proteins induced by this signaling pathway (Lee et al., 2003). This has also been supported in Nrf2 -/- mice, where treatment with the antioxidant tert-Butylhydroquinone (tBHQ) led to reduced glucuronidation activity in comparison with wild-type mice (Thimmulappa et al., 2002). In 2001, Enomoto and coworkers showed that administration of acetaminophen induced more severe centrilobular hepatocellular necrosis in Nrf2 null mice in comparison to wild-type mice. The authors suggested that the resulting sensitivity of Nrf2 -/- liver to electrophiles resulted from lack of cytoprotection due to decreased UGT and glutamate-cysteine ligase (GCL) expression (Enomoto et al., 2001). In 2007, Yueh et al. performed a series of experiments that resulted in the identification of an ARE sequence within the UGT1A1 promoter. HepG2 cells treated with the prooxidants tBHQ and BNF resulted in increased UGT1A1 glucuronidation. Loss of function analysis for Nrf2 conducted by siRNA targeted down regulation revealed that induction of UGT1A1 was not seen in Nrf2 deficient cells. Transgenic mice bearing the human UGT1 locus (TgUGT1) were treated with tBHQ to examine the contribution of oxidants toward the regulation of human UGT1A1 in vivo. UGT1A1 was significantly increased in liver and small and large intestines. Gene mapping experiments including transfections of UGT1A1 reporter gene constructs into HepG2 cells coupled with functional analysis of Nrf2 expression and binding to ARE ultimately confirmed the existence of an ARE in the phenobarbital-response enhancer module region of the UGT1A1 gene (Yueh and Tukey, 2007). To date, the list of

ARE-driven genes includes rat GSTA1, mouse GSTA1, rat GSTP1, rat NQO-1, human

NQO-1, human GCL, mouse ferritin-L, mouse metallothionein-1, multiple rat and mouse UGTs, and human UGT1A1 (Lee and Johnson, 2004; Yueh and Tukey, 2007).

The findings demonstrate a physiological role for Nrf2 in the UGT regulation.

The UGT1A1 Phenobarbital Response Enhancer Module

Increasing evidence has suggested that various xenobiotic receptors are capable of inducing a broad spectrum of hepatic and intestinal genes involved in xenobiotic metabolism (Qatanani and Moore, 2005; Ueda et al., 2002). In 2001,

Sugatani et al. reported that induction of UGT1A1 expression by PB could be credited to a core module in the distal UGT1A1 promoter region (Tolson and Wang, 2010).

This region was found to contain three putative nuclear receptor-binding motifs and was activated by CAR in co-transfected HepG2 cells as well as in mouse primary hepatocytes treated with PB (Sugatani et al., 2001). Following in 2003, Xie and colleagues demonstrated that UGT1A1 mRNA and protein were upregulated in both transgenic VP-hPXR mice and in rifampicin-treated, humanized PXR mice.

Subsequent in vitro experiments and electrophoretic mobility shift assays (EMSAs) revealed that the PXR/RXR heterodimer bound a nuclear receptor enhancer element in

UGT1A1 promoter region (Xie et al., 2003). Concurrently, independent studies also showed that the rodent-specific PXR agonist PCN increased UGT enzymatic activity and expression in wild-type, but not in Pxr -/- mice (Chen et al., 2003). Yueh et al. later showed that significant induction of UGT1A1 in HepG2 cells treated with prototypical AhR ligands, such as TCDD, BNF, and B[a]P metabolites was attributed

to a xenobiotic response element that was discovered by nucleotide sequence analysis of this UGT1A1 enhancer region (Yueh et al., 2003). Moreover, in 2007, Yueh et al. also documented UGT1A1 induction by antioxidants resulted directly from Nrf2 binding to an ARE sequence flanking the recently identified the AhR response element on the UGT1A1 gene (Yueh and Tukey, 2007). It is of significant interest that the CAR and PXR responsive elements, the AhR XRE, and the Nrf2 ARE are all located within the same 290-bp enhancer region, as this implies that the major xenobiotic responsive sequences of UGT1A1 tend to cluster together and may contribute to both induction efficacy and xenobiotic promiscuity (Tolson and Wang,

2010). This region of the UGT1A1 promoter that contains the enhancer sequences is widely known as the phenobarbital response enhancer module (PBREM). Today, the

PBREM has been determined to also include recognition sequences for the liver X receptor α (LXRα) (unpublished observations), PPARα (Senekeo-Effenberger et al.,

2007) and glucocorticoid receptor (Yueh et al., 2003; Sugatani et al., 2008; Sugatani et al., 2005; Usui et al., 2006). Mutations within this region significantly impact the enhancing effects of the classical UGT inducers, including chrysin, TCDD, B[a]P and

MC (Sugatani et al., 2004; Mackenzie et al., 2010), further verifying the importance of the PBREM in facilitating the extensive breadth of glucuronidation in xenobiotic metabolism.

The NF-κB/IKK Pathway

Addressing the signaling mechanisms involving other relevant transcription factors, such as NF-κB, is necessary for fully understanding how the DMEs are

regulated (Xu et al., 2005). In 1986, NF-κB was originally identified as a B-cell nuclear protein and named after its ability to bind to an intronic enhancer, termed the

κB motif, of the immunoglobulin κ-light chain gene (Sen and Baltimore, 2006; Sen and Baltimore, 1986). Activated NF-κB can bind these specific κB elements in target genes to regulate transcription of genes mediating inflammation, carcinogenesis and anti-apoptotic reactions (Chen et al., 2001). Numerous studies since have identified

NF-κB in several cell types and demonstrated activation by a wide range of inducers, including cytokines, mitogens, environmental and occupational particles or metals, intracellular stresses, viral and bacterial products, and UV light (Karin and Ben-

Neriah, 2000; Chen et al., 1999; Pahl, 1999; Gilmore, 1999; Sun and Ballard, 1999).

However, it was initially observed that NF-κB activity could be induced in the absence of new protein synthesis. This led to examination of the state of NF-κB in unstimulated 7OZ/3 cells by Baeuerle and Baltimore. Surprisingly, they observed little NF-κB activity in either the nucleus or cytoplasm of unstimulated cells.

Denaturation-renaturation of cytosolic fractions eventually revealed the presence of

NF-κB in cytoplasm, implicating that NF-κB was a cytoplasmic protein that was inhibited in its DNA-binding activity. The authors then utilized mild detergents, such as deoxycholate (DOC), to gently separate the inhibitor-NF-κB complex and were able to release cytosolic NF-κB from an inhibitory protein, IκB (Baeuerle and

Baltimore, 1988b). With the use of dissociating agents, they were also able to detect as much NF-κB in the cytosolic fraction from unstimulated 7OZ/3 cells as is found in the nuclear extract from phorbol ester-activated cells (Baeuerle and Baltimore, 1988a).

The discovery of this cytoplasmic inhibitor marked another milestone in NF-κB research and immediately increased interest in identifying the inhibitor and elucidating a physiologic mechanism for liberation of NF-κB from IκB (Hinz et al., 2012;

Kanarek and Ben-Neriah, 2012). It was later demonstrated that IκB exerted its inhibitory function by physically masking the NF-κB nuclear localization sequences

(Huxford et al., 1998; Beg et al., 1992; Henkel et al., 1992). Subsequent analysis of the IκB promoter and mRNA synthesis showed that transcription of IκB is regulated by NF-κB (Brown et al., 1993; de Martin et al., 1993; Sun et al., 1993).

IκBα is the most abundant NF-κB inhibitory protein (Chen et al., 1999).

Successive experiments in cell lines implicated early on that stimulus-induced IκB phosphorylation caused the release of NF-κB, which could account for activation of the transcription factor (Ghosh and Baltimore, 1990). The IκB kinase (IKK) complex, which consists of two catalytic subunits, IKKα and IKKβ, was determined to phosphorylate IκB (Karin and Ben-Neriah, 2000). However, it was eventually established that IκB phosphorylation was insufficient for NF-κB activation (Alkalay et al., 1995; DiDonato et al., 1995; Chen et al., 1995; Finco et al., 1994) and that IκB degradation preceded NF-κB activation (Brown et al., 1993; Sun et al., 1993; Beg et al., 1993; Mellits et al., 1993). In 1993, Henkel et al. investigated the fate of IκB after treatment with various NF-κB inducers, including phorbol ester, interleukin-1, lipopolysaccharide (LPS), and tumor necrosis factor-α (TNFα). The authors observed

IκB degradation after stimulation and that cell treatment with protease inhibitors or antioxidants completely inhibited IκB degradation and subsequent NF-κB activation,

thus confirming degradation of IκB as necessary for NF-κB activation (Henkel et al.,

1993). Further elucidation of the IκB proteolysis mechanism followed, first through the studies of Palombella et al., which demonstrated that proteasome inhibitor treatment abolished IκB degradation (Palombella et al., 1994). Soon after, several laboratories ascertained that signal-induced ubiquitination and proteasomal degradation of IκB was required for NF-κB activation (Chen et al., 1995; Palombella et al., 1994; Alkalay et al., 1995), implicating for the first time ubiquitin-dependent proteolysis as an integral step of signal-induced transcriptional activation (Kanarek and Ben-Neriah, 2012). Sequence homology comparisons and site-directed mutagenesis revealed a high degree of conservation in a short six amino acid N- terminal sequence of IκB. Among these, two conserved serine (Ser) residues were determined to be important in IκB phosphorylation. These residues were consistently phosphorylated post-phorbol ester simulation. In addition, signal-induced IκB ubiquitination, degradation, and NF-κB activation were abolished with their mutagenesis (Chen et al., 1995; Brown et al., 1995). In brief, inactive NF-κB is sequestered in the cytoplasm and bound to IκB, which masks NF-κB’s nuclear localization signal to prevent nuclear uptake. Upon cellular activation by extracellular stimuli, IκB is phosphorylated and targeted for rapid degradation. This results in NF-

κB release and translocation to the nucleus. Once within the nucleus, NF-κB can initiate or regulate early-response gene transcription by binding to κB motifs

(GGGRNNYYCC) located in the promoter or enhancer regions of target genes.

While NF-κB was originally studied for its role in activation of innate immune responses and inflammation, in 1996 it became clear that another very critical function

of NF-κB existed: inhibition of apoptosis (Liu et al., 1996; Li et al., 1999a; Wang et al., 1998; Beg and Baltimore, 1996). The first clue indicating this important function was directly observed from RelA knockout mice that died at mid-gestation from uncontrolled liver apoptosis (Li et al., 1999b). It was later determined that the extensive liver apoptosis in these mice was dependent on signaling via TNFR1 and that IKKβ- and IKKγ-deficient mice displayed similar yet more severe phenotypes (Li et al., 1999a; Makris et al., 2000). Over time, NF-κB has been documented as a transcriptional activator of many anti-apoptotic genes, including c-FLIP, c-IAP-1, c-

IAP-2, X chromosome-linked IAP (XIAP), Bcl-xL, Bcl-2 related protein A1 (A1/Bfl1),

Gadd45β, and SOD2 (Karin and Lin, 2002).

The NF-κB/IKK pathway has been shown to regulate transcription of important protective genes (Chen et al., 2001). As such, changes in transcriptional activation can result in alterations in target gene expression and thus contribute to toxicity or disease development. In 1995, studies demonstrated that infection and inflammatory diseases generally depressed CYP activity in rodents and humans

(Chang et al., 1978; Renton and Knickle, 1990). Moreover, in vivo and in vitro treatments with inflammatory stimuli, including LPS and cytokines, significantly decrease CYP2B and CYP3A expression and activity (Abdel-Razzak et al., 1995;

Beigneux et al., 2002; Li-Masters and Morgan, 2002; Muntane-Relat et al., 1995;

Pascussi et al., 2000; Sewer and Morgan, 1997). Similarly, human hepatocytes treated with IL-1β and LPS showed a down-regulation of UGT1A1, GSTA1, and GSTA2 expression (Assenat et al., 2004). Apigenin treatment of CaCo-2 cells by Svehlikova et al., found UGT1A1 induction to be associated with NF-κB translocation, as co-

treatment with the NF-κB nuclear translocation inhibitor, SN50, enhanced induction of

UGT1A1 by apigenin (Svehlikova et al., 2004). In contrast, neonatal hUGT1 mice that exhibit elevated serum bilirubin levels during development and have been treated orally with known activators of NF-κB, such as cadmium and LPS, (Hyun et al., 2007;

Luo et al., 2004), experience marked decreases in serum bilirubin and significant increases in intestinal UGT1A1 and Cyp2b10. An absence of induction of these genes was noted in the liver, implicating induction as intestinal specific. Fujiwara et al. consequently investigated the role of intestinal NF-κB in cadmium induction of

Cyp2b10 in a conditional knockout mouse model deficient in Ikkα/β specifically in intestinal epithelial cells (Fujiwara et al., 2012). Although these mice do not carry the

UGT1 transgene, evaluation of Cyp2b10 expression was still possible, since regulation occurs in a similar fashion to that of human UGT1A1 in hUGT1 mice. Induction of

Cyp2b10 expression was completely abolished in Vil-Cre/IkkαF/FIkkβF/F (GI knockout) mice treated with cadmium, compared to the cadmium-treated control IkkαF/F/IkkβF/F

(WT mice). These results suggest the existence of a close relationship between NF-κB expression in intestinal cells and expression of UGT1A1.

The Mitogen Activated Protein Kinases

Addressing the signal transduction cascades, such as the Mitogen Activated

Protein Kinases (MAPKs), in addition to the signaling mechanism involving the xenobiotic receptors and other relevant transcription factors, is required to fully understand DME regulation (Xu et al., 2005). The discovery of the MAPKs occurred approximately 20 years ago, as evidence began to build that suggested insulin and

mitogens acted through novel, possibly similar mechanisms to promote intracellular protein phosphorylation (Avruch et al., 1982; Blackshear et al., 1983) through the activation of protein Ser/threonine (Thr) kinases (Cobb and Rosen, 1983; Novak-

Hofer and Thomas, 1984). Sturgill and Ray were the first to detect insulin-activated protein Ser/Thr kinase activity in extracts of 3T3-L1 adipocytes that were capable of phosphorylating a contaminating high molecular weight polypeptide identified as microtubule-associated protein-2 (MAP-2) (Sturgill and Ray, 1986). Insulin activation of this partially purified kinase was accompanied by an increase in 32P incorporation onto its tyrosine (Tyr) and Thr residues (Ray and Sturgill, 1988a). Anti- phospho-Tyr antibodies also adsorbed kinase activity, confirming that Tyr phosphorylation of the kinase polypeptide occurred with its activation (Ray and

Sturgill, 1988b). In addition, in vitro treatment with either a Tyr-specific or a Ser/Thr- specific protein phosphatase resulted in the deactivation of the kinase (Anderson et al.,

1990). At the time, few other regulatory Tyr phosphorylations had been identified apart from those on various receptor and nonreceptor Tyr kinases themselves (Hunter and Cooper, 1985). Furthermore, the absolute increase in Ser/Thr phosphorylation of cellular proteins exceeded the increase in Tyr phosphorylation observed in cells expressing constitutively active Tyr kinases by 100-1000 fold (Cooper and Hunter,

1981). The possibility that this MAP-2 kinase might be a ubiquitous effector of mitogenic stimuli was further supported by the finding that the MAP-2 kinase polypeptide was identical to previously characterized 41-43 kDa polypeptides whose

Tyr phosphorylation was stimulated by several growth factors and active phorbol esters (Rossomando et al., 1989; Nakamura et al., 1983; Cooper et al., 1984; Cooper

and Hunter, 1985; Kohno, 1985; Gilmore and Martin, 1983; Bishop et al., 1983).

These findings ultimately fueled the renaming of the acronym “MAP” from

“microtubule-associated protein” to “mitogen-activated protein”, as we know it today.

However, the MAP kinase (MAPK) was not the first insulin-mitogen activated protein

(Ser/Thr) kinase described. Earlier work from several labs had shown that in vivo

Ser/Thr phosphorylation of the ribosomal protein S6 in response to insulin or mitogen stimulation (Gressner and Wool, 1976; Haselbacher et al., 1979; Smith et al., 1979) was paralleled by the appearance of stably activated, 40S-S6 selective protein kinase activities in stimulated cell extracts (Cobb and Rosen, 1983; Novak-Hofer and

Thomas, 1984). The first of these S6 kinases (now called Rsks) was purified from

Xenopus oocytes (Erikson and Maller, 1985; Erikson and Maller, 1986). Direct microinjection into oocytes of Tyr kinase polypeptides, such as vSrc, vAbl, and the insulin receptor itself provided evidence that these S6 kinases were activated downstream of Tyr kinases (Spivack et al., 1984; Maller et al., 1985; Stefanovic et al.,

1986). Activation of the Xenopus S6 kinase was determined to occur exclusively through Ser/Thr phosphorylation, since the insulin-activated S6 kinase lacked incorporation of 32P at its Tyr residue and its activity was eliminated by Ser/Thr- specific phosphatases (Maller, 1986). Amazingly, it was then found that the partially purified, insulin-activated MAPK also directly phosphorylated and activated the purified Xenopus S6 kinase (Sturgill et al., 1988). Independent studies had concurrently identified a set of mitogen activated S6 peptide kinases in extracts of

EGF-treated NIH3T3 cells that were activated by upstream, EGF-regulated kinases

(Ahn et al., 1990; Ahn and Krebs, 1990; Ahn et al., 1991). The ability of the MAPK

to activate an S6 kinase identified what proved to be the first physiologic MAPK substrate, marking an important milestone in growth factor signaling (Avruch, 2007).

However, the concept of a protein kinase cascade was not particularly novel. Krebs and colleagues were responsible for defining the first example, in the activation of phosphorylase b kinase by the cyclic AMP-dependent protein kinase (Walsh et al.,

1968). It was also widely recognized that phosphorylation by an upstream kinase was required for AMP-activated protein kinase activity (Ingebritsen et al., 1978). Then in

1990, a surprising aspect of the MAPK cascade was revealed via the molecular cloning of a cDNA encoding the MAPK polypeptide (Boulton et al., 1990). It was found that the primary sequence of the p44 MAPK (designated as extracellular signal- related kinase 1(ERK1)) was nearly 50% identical to the sequences of two recently described S. cerevisiae protein kinases, KSS1 and FUS3, that had been identified as participants in the yeast mating pathway (Courchesne et al., 1989; Elion et al., 1990).

The remarkably high conservation of structure across an enormous phyllogenetic distance indicates that the role of this family of protein kinases as mediators of receptor-regulated cellular differentiation and proliferation is both ancient and highly conserved (Avruch, 2007).

These findings were followed by an intense effort to define the order and biochemical actions of each of these yeast kinases, as well as the identity of the upstream activators of the MAPKs evident in various vertebrate systems (Avruch,

2007). In the 1990s, there were a series of reports of a partially purified cytoplasmic protein (50-60 kDa) that functioned as a MAPK activator. This activator was capable of promoting in vitro phosphorylation of MAPKs, ERK1 and ERK2, at both Thr and

Tyr residues, which resulted in increased MAPK activity (Ahn et al., 1991; Gomez and Cohen, 1991; Ahn et al., 1992). These findings eliminated the possibility that the

MAPK was simply the direct substrate of a Tyr-specific kinase. The discovery that the MAPKs auto-activated slowly in vitro by auto-phosphorylation and of the inability of MAPK activators to catalyze significant phosphorylation of other polypeptides led to great uncertainty as to whether the MAPK activators were actually protein kinases

(Avruch, 2007). However, this issue was eventually settled with the studies that demonstrated that MAPK activators could phosphorylate catalytically-inactive mutant

MAPK polypeptides. These activators are now denoted as MAPK kinases (MAP2Ks) or MAP and ERK kinases (MEKs). Analysis of primary MAP2K sequences from various sources revealed that the vertebrate MAP2Ks were 30-40% identical in overall primary sequence to one of the essential protein kinases of the S. cerevisiae mating pathway, STE7 (Nakielny et al., 1992; Crews and Erikson, 1992; Kosako et al., 1992;

Wu et al., 1992; Crews et al., 1992; Seger et al., 1992), and consequently two kinases,

MKK1 and MKK2, each capable of activating ERK1 and ERK2, were identified.

Over time, subsequent studies led to the identification of MAP2K upstream kinase regulators (MAP3Ks and MAP4Ks) as well as the discovery of the two additional mammalian MAPK subfamilies, stress-activated protein kinase (SAPK)/ c-Jun NH2- terminal protein kinase (JNK) (Derijard et al., 1994; Kyriakis et al., 1994) and p38

(Han et al., 1994; Rouse et al., 1994; Freshney et al., 1994; Lee et al., 1994). It was eventually determined that MAPKs were activated by dual phosphorylation of a tripeptide motif (Thr-Xaa-Tyr) located in the activation loop (T-loop). This phosphorylation is mediated by a MAP2K, which is activated by phosphorylation by a

MAP3K, thus demonstrating that MAPK activation occurs via a kinase signaling cascade. These exciting discoveries that unveiled the MAPK pathway in combination with identifying MAP3K/MAP2K/MAPK cassettes in S. cerevisiae and S. pombe that were distinct from their mating pathways (Levin and Errede, 1995), provided a forceful demonstration that evolution conserved not only critical housekeeping molecules, but useful regulatory modules and designs as well. Today, it is well established that MAPK cascades are evolutionarily conserved in all eukaryotes and play a key role in the regulation of gene expression (Schaeffer and Weber, 1999).

It has recently been shown through transient transfection studies in HepG2 cells as well as treatment with kinase inhibitors that induction of ARE-dependent

Phase II detoxifying enzymes is mediated by a MAPK pathway (Yu et al., 2000;

Keum et al., 2003; Shen et al., 2004; Yu et al., 1999). Specific to UGT gene regulation, investigation of the mechanism involved in UGT1A1 induction by the important dietary flavonoid chrysin in cell culture identified a previously unknown mechanism of UGT1A1 regulation through the MAPK pathway. siRNA down regulation of AhR, a typical mediator of UGT1A1 expression, revealed that chrysin utilized AhR in conjunction with other factors through MAPK signaling pathways to maximally induce UGT1A1. Treatment with various MAPK inhibitors has also been observed to suppress chrysin induction of UGT1A1 luciferase activity (Bonzo et al.,

2007). Moreover, induction of UGT1A1 transcription by sulforaphane has also been linked to the MAPKs, as induction was completely abolished by PD98059, a selective inhibitor of MEK1, which is an upstream kinase regulating ERK1/2 (Svehlikova et al.,

2004). These findings demonstrate that the MAPKs can act as upstream activators of

transcription factors that regulate important DMEs.

Objectives of the Dissertation

I have been very lucky to have such a well-rounded research experience throughout graduate school. My work has exposed me to two very important, yet separate areas of focus of the UGT superfamily of enzymes: protein structure and gene regulation. I investigated the functional relevance of UGT intermolecular interactions in tissue culture, utilizing human hepatocytes and siRNA technology, and I studied

UGT1A1 gene regulation by oral arsenic in a humanized mouse model system. Both projects emphasize the inherent complexities of the UGTs, as activity and subsequent drug metabolism and disposition can be affected by several factors, including dimerization, heredity, and environmental toxicant exposure.

When I officially joined the Tukey lab at the beginning of graduate school,

one of the doctoral candidates (Dr. Theresa Operaña) was focusing the majority of

her thesis work on intermolecular interactions of the UGTs through the use of FRET

technology in combination with co-immunoprecipitation experiments. Her co-

expression studies in COS cells revealed that all the UGT1A proteins homo-

dimerized and that UGT1A1 hetero-dimerized with UGT1A3, UGT1A4, UGT1A6,

UGT1A7, UGT1A8, UGT1A9, and UGT1A10. Additionally, co-expression of

UGT1A1 and UGT1A7 increased 2-napthol glucuronidation in comparison to cells

only expressing either UGT1A1 or UGT1A7, suggesting that protein interactions

could impact enzyme function (Operaña and Tukey, 2007). This aspect of her work

had always interested me and during the summer of 2011, I pursued an opportunity

to further investigate UGT-UGT interactions as a graduate intern in the PKDM

department at Amgen, Inc. With the abundance of in vitro data supporting the

functional relevance of UGT dimerization, I became interested in addressing whether

dimerization was capable of modulating enzyme functionality in biologically active

systems, such as human hepatocytes. To test my hypothesis, I utilized selective

siRNA down regulation experiments and activity studies to examine changes in

metabolite formation due to disrupted protein interactions. Through my studies, I

was able to demonstrate siRNA down regulation as an important process for

evaluating UGT enzymology and that UGT-UGT interactions are a physiologically

relevant phenomena whose effects can be observed in human hepatocytes.

Therefore, human hepatocytes can potentially serve as a more reliable method for

characterizing UGTs and phenotyping UGT substrates.

The advent of transgenic technologies in the 1980s opened many doors in medical and research fields (Dunn et al., 2005; Gordon and Ruddle, 1981). This has been particularly the case for the study of UGT1 gene regulation in vivo and understanding human UGT1A1 deficiencies. In 2010, our laboratory successfully generated a fully humanized mouse model (hUGT1) by crossing TgUGT1 mice expressing the entire human UGT1 locus with Ugt1-null mice. While Ugt1-null mice died within seven days after birth due to a UGT1A1 glucuronidation deficiency, the humanized mice did not exhibit the same developmental lethality (Fujiwara et al.,

2010b). Further characterization of the UGT1 humanized model revealed that during development, neonatal bilirubin levels rapidly increased, peaking at around 14 days after birth and then decreased to normal adult levels around 21 days. The observed

rise and fall in bilirubin levels was determined to be directly due to developmental expression of small intestinal UGT1A1 (Fujiwara et al., 2010b).

Since numerous environmental toxicants induce UGT1A1 through association with xenobiotic and environmental receptors, I speculated that induction of the

UGT1A1 gene by environmental contaminants might alter serum bilirubin levels.

Earlier work in our lab had already demonstrated that treatment of the hUGT1 mice with UGT1A1-inducing agents, such as PB and TCDD, led to a significant reduction in serum bilirubin. With UGT1A1 gene expression being controlled and regulated in vivo by inducible and tissue specific factors, I decided to explore the association between exposure to prominent metal contaminants and gene expression through fluctuations in bilirubin levels.

Environmental arsenic contamination is a significant problem worldwide, with drinking water being the most common source of arsenic exposure. There is extensive epidemiological evidence linking arsenic exposure to various diseases

(arteriosclerosis, cardiovascular disease, Blackfoot disease) (Navas-Acien et al.,

2005), numerous cancers (skin, bladder, lung, liver, prostate, kidney) (Kligerman and

Tennant, 2007; Chen et al., 1992), diabetes (Diaz-Villasenor et al., 2007) and certain neurological ailments (Alzheimer’s and Parkinson’s) (Schmuck et al., 2005; Vahidnia et al., 2007), which suggests that arsenic has a very extensive effect and is capable of altering various signaling pathways. Therefore, understanding the development of toxicities associated with arsenic exposure is complicated.

With respect to the field of drug metabolism, there is very little information on how arsenic exposure modulates UGT1 gene expression. This is of great concern,

considering how prominent arsenic exposure has recently become in the news. The shocking and on-going prevalence of contamination led me to question whether arsenic was capable of affecting important biological processes, such as drug metabolism. More specifically, I wanted to investigate the effects of oral arsenic exposure on UGT1A1 expression in our hUGT1 mouse model. Treatment of neonatal hUGT1 mice with arsenic caused decreases in serum bilirubin and significant induction of small intestinal UGT1A1. This then led me to further investigate the mechanism by which induction was occurring.

Many of the toxic effects of arsenic exposure can be attributed to an assortment of cellular responses, including altered nuclear transcription factor activities or signal transduction, inflammation, and oxidative stress, which led to the investigation of nuclear receptors (Kaltreider et al., 2001; Bonzo et al., 2005; Noreault et al., 2005;

Medina-Diaz et al., 2009; Baldwin and Roling, 2009), NF-κB (Kapahi et al., 2000;

Roussel and Barchowsky, 2000), and Nrf2 (Pi et al., 2003; Kumagai and Sumi, 2007;

De Vizcaya-Ruiz et al., 2009) in regulating intestinal UGT1A1 induction. However, the contribution of arsenic to such a variety of different diseases also indicated to me that it might not function through one specific mechanism, but instead by eliciting a more global effect. Both abnormal cell cycle regulation (Bonzo et al., 2005; Lau et al., 2004; Eguchi et al., 2011) and changes in cellular morphology due to arsenic- induced cytotoxicity (Yancy et al., 2005; Li et al., 2011) have been observed with exposure and can lead to changes in signaling that ultimately affect gene expression.

This directed my attention towards immunohistochemical assessment of intestinal damage, changes in cellular morphology, and alterations in cellular proliferation.

These studies collectively led to the identification of a novel mechanism that consists of three potential pathways by which oral arsenic modulates small intestinal UGT1A1 expression in hUGT1 mice.

References

Abdel-Razzak Z, Corcos L, Fautrel A and Guillouzo A (1995) Interleukin-1 beta antagonizes phenobarbital induction of several major cytochromes P450 in adult rat hepatocytes in primary culture. FEBS letters 366:159-164.

Ahn NG and Krebs EG (1990) Evidence for an epidermal growth factor-stimulated protein kinase cascade in Swiss 3T3 cells. Activation of serine peptide kinase activity by myelin basic protein kinases in vitro. The Journal of biological chemistry 265:11495-11501.

Ahn NG, Seger R, Bratlien RL, Diltz CD, Tonks NK and Krebs EG (1991) Multiple components in an epidermal growth factor-stimulated protein kinase cascade. In vitro activation of a myelin basic protein/microtubule-associated protein 2 kinase. The Journal of biological chemistry 266:4220-4227.

Ahn NG, Seger R and Krebs EG (1992) The mitogen-activated protein kinase activator. Current opinion in cell biology 4:992-999.

Ahn NG, Weiel JE, Chan CP and Krebs EG (1990) Identification of multiple epidermal growth factor-stimulated protein serine/threonine kinases from Swiss 3T3 cells. The Journal of biological chemistry 265:11487-11494.

Alkalay I, Yaron A, Hatzubai A, Jung S, Avraham A, Gerlitz O, Pashut-Lavon I and Ben-Neriah Y (1995) In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B. Molecular and cellular biology 15:1294-1301.

Allen SW, Mueller L, Williams SN, Quattrochi LC and Raucy J (2001) The use of a high-volume screening procedure to assess the effects of dietary flavonoids on human cyp1a1 expression. Drug metabolism and disposition: the biological fate of chemicals 29:1074-1079.

Alves R, Chaleil RA and Sternberg MJ (2002) Evolution of enzymes in metabolism: a network perspective. Journal of molecular biology 320:751-770.

Anderson NG, Maller JL, Tonks NK and Sturgill TW (1990) Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343:651-653.

Andrews NC, Erdjument-Bromage H, Davidson MB, Tempst P and Orkin SH (1993) Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein. Nature 362:722-728.

Arias IM (1962) Chronic unconjugated hyperbilirubinemia without overt signs of hemolysis in adolescents and adults. The Journal of clinical investigation 41:2233- 2245.

Arias IM, Gartner LM, Cohen M, Ezzer JB and Levi AJ (1969) Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency. Clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. The American journal of medicine 47:395-409.

Assenat E, Gerbal-Chaloin S, Larrey D, Saric J, Fabre JM, Maurel P, Vilarem MJ and Pascussi JM (2004) Interleukin 1beta inhibits CAR-induced expression of hepatic genes involved in drug and bilirubin clearance. Hepatology 40:951-960.

Avruch J (2007) MAP kinase pathways: the first twenty years. Biochimica et biophysica acta 1773:1150-1160.

Avruch J, Alexander MC, Palmer JL, Pierce MW, Nemenoff RA, Blackshear PJ, Tipper JP and Witters LA (1982) Role of insulin-stimulated protein phosphorylation in insulin action. Federation proceedings 41:2629-2633.

Axelrod J, Kalckar HM, Maxwell ES and Strominger JL (1957) Enzymatic formation of uridine diphosphoglucuronic acid. The Journal of biological chemistry 224:79-90.

Bachmann K, Patel H, Batayneh Z, Slama J, White D, Posey J, Ekins S, Gold D and Sambucetti L (2004) PXR and the regulation of apoA1 and HDL-cholesterol in rodents. Pharmacological research : the official journal of the Italian Pharmacological Society 50:237-246.

Baes M, Gulick T, Choi HS, Martinoli MG, Simha D and Moore DD (1994) A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements. Molecular and cellular biology 14:1544- 1552.

Baeuerle PA and Baltimore D (1988a) Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell 53:211- 217.

Baeuerle PA and Baltimore D (1988b) I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science 242:540-546.

Bailey A, Robinson D and Dawson AM (1977) Does Gilbert's disease exist? Lancet 1:931-933.

Baldwin WS and Roling JA (2009) A concentration addition model for the activation of the constitutive androstane receptor by xenobiotic mixtures. Toxicological sciences : an official journal of the Society of Toxicology 107:93-105.

Ballario P, Talora C, Galli D, Linden H and Macino G (1998) Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospora crassa white collar proteins. Molecular microbiology 29:719-729.

Baulcombe D (1999) Viruses and gene silencing in plants. Archives of virology Supplementum 15:189-201.

Beato M (1989) Gene regulation by steroid hormones. Cell 56:335-344.

Beato M (1991) Transcriptional control by nuclear receptors. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 5:2044- 2051.

Beaulieu M, Levesque E, Hum DW and Belanger A (1996) Isolation and characterization of a novel cDNA encoding a human UDP-glucuronosyltransferase active on C19 steroids. The Journal of biological chemistry 271:22855-22862.

Beaulieu M, Levesque E, Hum DW and Belanger A (1998) Isolation and characterization of a human orphan UDP-glucuronosyltransferase, UGT2B11. Biochemical and biophysical research communications 248:44-50.

Beaulieu M, Levesque E, Tchernof A, Beatty BG, Belanger A and Hum DW (1997) Chromosomal localization, structure, and regulation of the UGT2B17 gene, encoding a C19 steroid metabolizing enzyme. DNA and cell biology 16:1143-1154.

Beg AA and Baltimore D (1996) An essential role for NF-kappaB in preventing TNF- alpha-induced cell death. Science 274:782-784.

Beg AA, Finco TS, Nantermet PV and Baldwin AS, Jr. (1993) Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Molecular and cellular biology 13:3301-3310.

Beg AA, Ruben SM, Scheinman RI, Haskill S, Rosen CA and Baldwin AS, Jr. (1992) I kappa B interacts with the nuclear localization sequences of the subunits of NF- kappa B: a mechanism for cytoplasmic retention. Genes & development 6:1899-1913.

Beigneux AP, Moser AH, Shigenaga JK, Grunfeld C and Feingold KR (2002) Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response. Biochemical and biophysical research communications 293:145-149.

Belanger A, Hum DW, Beaulieu M, Levesque E, Guillemette C, Tchernof A, Belanger G, Turgeon D and Dubois S (1998) Characterization and regulation of UDP- glucuronosyltransferases in steroid target tissues. The Journal of steroid biochemistry and molecular biology 65:301-310.

Berg JM (1989) DNA binding specificity of steroid receptors. Cell 57:1065-1068.

Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, Ohlsson R, Postlind H, Blomquist P and Berkenstam A (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proceedings of the National Academy of Sciences of the United States of America 95:12208-12213.

Bhalla S, Ozalp C, Fang S, Xiang L and Kemper JK (2004) Ligand-activated pregnane X receptor interferes with HNF-4 signaling by targeting a common coactivator PGC- 1alpha. Functional implications in hepatic cholesterol and glucose metabolism. The Journal of biological chemistry 279:45139-45147.

Billing BH, Cole PG and Lathe GH (1957) The excretion of bilirubin as a diglucuronide giving the direct van den Bergh reaction. The Biochemical journal 65:774-784.

Bishop R, Martinez R, Nakamura KD and Weber MJ (1983) A tumor promoter stimulates phosphorylation on tyrosine. Biochemical and biophysical research communications 115:536-543.

Blackshear PJ, Nemenoff RA and Avruch J (1983) Insulin and growth factors stimulate the phosphorylation of a Mr-22000 protein in 3T3-L1 adipocytes. The Biochemical journal 214:11-19.

Blumberg B, Sabbagh W, Jr., Juguilon H, Bolado J, Jr., van Meter CM, Ong ES and Evans RM (1998) SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes & development 12:3195-3205.

Bock KW (2003) Vertebrate UDP-glucuronosyltransferases: functional and evolutionary aspects. Biochemical pharmacology 66:691-696.

Bock KW and Kohle C (2009) Topological aspects of oligomeric UDP- glucuronosyltransferases in endoplasmic reticulum membranes: advances and open questions. Biochemical pharmacology 77:1458-1465.

Bock KW, Raschko FT, Gschaidmeier H, Seidel A, Oesch F, Grove AD and Ritter JK (1999) Mono- and Diglucuronide formation from benzo[a]pyrene and chrysene diphenols by AHH-1 cell-expressed UDP-glucuronosyltransferase UGT1A7. Biochemical pharmacology 57:653-656.

Bock KW, von Clausbruch UC, Josting D and Ottenwalder H (1977) Separation and partial purification of two differentially inducible UDP-glucuronyltransferases from rat liver. Biochemical pharmacology 26:1097-1100.

Bonzo JA, Belanger A and Tukey RH (2007) The role of chrysin and the ah receptor in induction of the human UGT1A1 gene in vitro and in transgenic UGT1 mice. Hepatology 45:349-360.

Bonzo JA, Chen S, Galijatovic A and Tukey RH (2005) Arsenite inhibition of CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin is independent of cell cycle arrest. Molecular pharmacology 67:1247-1256.

Bosma PJ, Chowdhury JR, Bakker C, Gantla S, de Boer A, Oostra BA, Lindhout D, Tytgat GN, Jansen PL, Oude Elferink RP and et al. (1995) The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. The New England journal of medicine 333:1171-1175.

Bosma PJ, Goldhoorn B, Oude Elferink RP, Sinaasappel M, Oostra BA and Jansen PL (1993) A mutation in bilirubin uridine 5'-diphosphate-glucuronosyltransferase isoform 1 causing Crigler-Najjar syndrome type II. Gastroenterology 105:216-220.

Bosma PJ, Seppen J, Goldhoorn B, Bakker C, Oude Elferink RP, Chowdhury JR, Chowdhury NR and Jansen PL (1994) Bilirubin UDP-glucuronosyltransferase 1 is the only relevant bilirubin glucuronidating isoform in man. The Journal of biological chemistry 269:17960-17964.

Boulton TG, Yancopoulos GD, Gregory JS, Slaughter C, Moomaw C, Hsu J and Cobb MH (1990) An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 249:64-67.

Brown K, Gerstberger S, Carlson L, Franzoso G and Siebenlist U (1995) Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science 267:1485-1488.

Brown K, Park S, Kanno T, Franzoso G and Siebenlist U (1993) Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. Proceedings of the National Academy of Sciences of the United States of America 90:2532-2536.

Buckley DB and Klaassen CD (2009) Induction of mouse UDP- glucuronosyltransferase mRNA expression in liver and intestine by activators of arylhydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, peroxisome proliferator-activated receptor alpha, and nuclear factor erythroid 2-related factor 2. Drug metabolism and disposition: the biological fate of chemicals 37:847- 856.

Burbach KM, Poland A and Bradfield CA (1992) Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. Proceedings of the National Academy of Sciences of the United States of America 89:8185-8189.

Burchell B (1978) Substrate specificity and properties of uridine diphosphate glucuronyltransferase purified to apparent homogeneity from phenobarbital-treated rat liver. The Biochemical journal 173:749-757.

Burchell B and Coughtrie MW (1989) UDP-glucuronosyltransferases. Pharmacology & therapeutics 43:261-289.

Burchell B, Nebert DW, Nelson DR, Bock KW, Iyanagi T, Jansen PL, Lancet D, Mulder GJ, Chowdhury JR, Siest G and et al. (1991) The UDP glucuronosyltransferase gene superfamily: suggested nomenclature based on evolutionary divergence. DNA and cell biology 10:487-494.

Burchell B, Soars M, Monaghan G, Cassidy A, Smith D and Ethell B (2000) Drug- mediated toxicity caused by genetic deficiency of UDP-glucuronosyltransferases. Toxicology letters 112-113:333-340.

Carlstedt-Duke J, Okret S, Wrange O and Gustafsson JA (1982) Immunochemical analysis of the glucocorticoid receptor: identification of a third domain separate from the steroid-binding and DNA-binding domains. Proceedings of the National Academy of Sciences of the United States of America 79:4260-4264.

Carlstedt-Duke J, Stromstedt PE, Persson B, Cederlund E, Gustafsson JA and Jornvall H (1988) Identification of hormone-interacting amino acid residues within the steroid- binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors. The Journal of biological chemistry 263:6842-6846.

Carlstedt-Duke J, Stromstedt PE, Wrange O, Bergman T, Gustafsson JA and Jornvall H (1987) Domain structure of the glucocorticoid receptor protein. Proceedings of the National Academy of Sciences of the United States of America 84:4437-4440.

Chan JY, Han XL and Kan YW (1993) Isolation of cDNA encoding the human NF-E2 protein. Proceedings of the National Academy of Sciences of the United States of America 90:11366-11370.

Chan K and Kan YW (1999) Nrf2 is essential for protection against acute pulmonary injury in mice. Proceedings of the National Academy of Sciences of the United States of America 96:12731-12736.

Chan K, Lu R, Chang JC and Kan YW (1996) NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proceedings of the National Academy of Sciences of the United States of America 93:13943-13948.

Chanas SA, Jiang Q, McMahon M, McWalter GK, McLellan LI, Elcombe CR, Henderson CJ, Wolf CR, Moffat GJ, Itoh K, Yamamoto M and Hayes JD (2002) Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. The Biochemical journal 365:405- 416.

Chang JC, Liu D and Kan YW (1992) A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression. Proceedings of the National Academy of Sciences of the United States of America 89:3107-3110.

Chang KC, Bell TD, Lauer BA and Chai H (1978) Altered theophylline pharmacokinetics during acute respiratory viral illness. Lancet 1:1132-1133.

Chawla A, Repa JJ, Evans RM and Mangelsdorf DJ (2001) Nuclear receptors and lipid physiology: opening the X-files. Science 294:1866-1870.

Chen C, Staudinger JL and Klaassen CD (2003) Nuclear receptor, pregname X receptor, is required for induction of UDP-glucuronosyltranferases in mouse liver by pregnenolone-16 alpha-carbonitrile. Drug metabolism and disposition: the biological fate of chemicals 31:908-915.

Chen CJ, Chen CW, Wu MM and Kuo TL (1992) Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. British journal of cancer 66:888-892.

Chen F, Castranova V, Shi X and Demers LM (1999) New insights into the role of nuclear factor-kappaB, a ubiquitous transcription factor in the initiation of diseases. Clinical chemistry 45:7-17.

Chen F, Ding M, Castranova V and Shi X (2001) Carcinogenic metals and NF-kappaB activation. Molecular and cellular biochemistry 222:159-171.

Chen F, Ritter JK, Wang MG, McBride OW, Lubet RA and Owens IS (1993) Characterization of a cloned human dihydrotestosterone/androstanediol UDP- glucuronosyltransferase and its comparison to other steroid isoforms. Biochemistry 32:10648-10657.

Chen S, Beaton D, Nguyen N, Senekeo-Effenberger K, Brace-Sinnokrak E, Argikar U, Remmel RP, Trottier J, Barbier O, Ritter JK and Tukey RH (2005) Tissue-specific, inducible, and hormonal control of the human UDP-glucuronosyltransferase-1 (UGT1) locus. The Journal of biological chemistry 280:37547-37557.

Chen YH and Tukey RH (1996) Protein kinase C modulates regulation of the CYP1A1 gene by the aryl hydrocarbon receptor. The Journal of biological chemistry 271:26261-26266.

Chen Z, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D and Maniatis T (1995) Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes & development 9:1586-1597.

Chirulli V, Longo V, Marini S, Mazzaccaro A, Fiorio R and Gervasi PG (2005) CAR and PXR expression and inducibility of CYP2B and CYP3A activities in rat and rabbit lungs. Life sciences 76:2535-2546.

Cho HY, Jedlicka AE, Reddy SP, Kensler TW, Yamamoto M, Zhang LY and Kleeberger SR (2002) Role of NRF2 in protection against hyperoxic lung injury in mice. American journal of respiratory cell and molecular biology 26:175-182.

Cho JY, Kang DW, Ma X, Ahn SH, Krausz KW, Luecke H, Idle JR and Gonzalez FJ (2009) Metabolomics reveals a novel vitamin E metabolite and attenuated vitamin E metabolism upon PXR activation. Journal of lipid research 50:924-937.

Choi HS, Chung M, Tzameli I, Simha D, Lee YK, Seol W and Moore DD (1997) Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR. The Journal of biological chemistry 272:23565-23571.

Ciotti M, Chen F, Rubaltelli FF and Owens IS (1998) Coding defect and a TATA box mutation at the bilirubin UDP-glucuronosyltransferase gene cause Crigler-Najjar type I disease. Biochimica et biophysica acta 1407:40-50.

Clever U and Karlson P (1960) [Induction of puff changes in the salivary gland chromosomes of Chironomus tentans by ecdysone]. Experimental cell research 20:623-626.

Cobb MH and Rosen OM (1983) Description of a protein kinase derived from insulin- treated 3T3-L1 cells that catalyzes the phosphorylation of ribosomal protein S6 and casein. The Journal of biological chemistry 258:12472-12481.

Coffman BL, Rios GR, King CD and Tephly TR (1997) Human UGT2B7 catalyzes morphine glucuronidation. Drug metabolism and disposition: the biological fate of chemicals 25:1-4.

Cogoni C, Irelan JT, Schumacher M, Schmidhauser TJ, Selker EU and Macino G (1996) Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. The EMBO journal 15:3153-3163.

Cogoni C and Macino G (1999a) Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399:166-169.

Cogoni C and Macino G (1999b) Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase. Science 286:2342-2344.

Collis P, Antoniou M and Grosveld F (1990) Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression. The EMBO journal 9:233-240.

Conti A and Bickel MH (1977) History of Drug Metabolism: Discoveries of the Major Pathways in the 19th Century. Drug metabolism reviews 6:1-50.

Cooper JA and Hunter T (1981) Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Molecular and cellular biology 1:165-178.

Cooper JA and Hunter T (1985) Major substrate for growth factor-activated protein- tyrosine kinases is a low-abundance protein. Molecular and cellular biology 5:3304- 3309.

Cooper JA, Sefton BM and Hunter T (1984) Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells. Molecular and cellular biology 4:30-37.

Courchesne WE, Kunisawa R and Thorner J (1989) A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae. Cell 58:1107- 1119.

Coveney PV, Swadling JB, Wattis JA and Greenwell HC (2012) Theory, modelling and simulation in origins of life studies. Chemical Society reviews.

Crews CM, Alessandrini A and Erikson RL (1992) The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science 258:478-480.

Crews CM and Erikson RL (1992) Purification of a murine protein-tyrosine/threonine kinase that phosphorylates and activates the Erk-1 gene product: relationship to the fission yeast byr1 gene product. Proceedings of the National Academy of Sciences of the United States of America 89:8205-8209.

Crews ST (1998) Control of cell lineage-specific development and transcription by bHLH-PAS proteins. Genes & development 12:607-620.

Crigler JF, Jr. and Najjar VA (1952a) Congenital familial nonhemolytic jaundice with kernicterus. Pediatrics 10:169-180.

Crigler JF, Jr. and Najjar VA (1952b) Congenital familial nonhemolytic jaundice with kernicterus; a new clinical entity. AMA American journal of diseases of children 83:259-260.

Crosthwaite SK, Dunlap JC and Loros JJ (1997) Neurospora wc-1 and wc-2: transcription, photoresponses, and the origins of circadian rhythmicity. Science 276:763-769.

Cui Y, Konig J, Leier I, Buchholz U and Keppler D (2001) Hepatic uptake of bilirubin and its conjugates by the human organic anion transporter SLC21A6. The Journal of biological chemistry 276:9626-9630.

Davies NM, Teng XW and Skjodt NM (2003) Pharmacokinetics of rofecoxib: a specific cyclo-oxygenase-2 inhibitor. Clinical pharmacokinetics 42:545-556.

Davis RL, Cheng PF, Lassar AB and Weintraub H (1990) The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. Cell 60:733- 746. de Martin R, Vanhove B, Cheng Q, Hofer E, Csizmadia V, Winkler H and Bach FH (1993) Cytokine-inducible expression in endothelial cells of an I kappa B alpha-like gene is regulated by NF kappa B. The EMBO journal 12:2773-2779. de Morais SM, Chow SY and Wells PG (1992) Biotransformation and toxicity of acetaminophen in congenic RHA rats with or without a hereditary deficiency in bilirubin UDP-glucuronosyltransferase. Toxicology and applied pharmacology 117:81-87.

De Vizcaya-Ruiz A, Barbier O, Ruiz-Ramos R and Cebrian ME (2009) Biomarkers of oxidative stress and damage in human populations exposed to arsenic. Mutation research 674:85-92.

Del Villar E, Sanchez E, Autor AP and Tephly TR (1975) Morphine metabolism. III. Solubilization and separation of morphine and p-nitrophenol uridine diphosphoglucuronyltransferases. Molecular pharmacology 11:236-240.

Denis M, Cuthill S, Wikstrom AC, Poellinger L and Gustafsson JA (1988) Association of the dioxin receptor with the Mr 90,000 heat shock protein: a structural kinship with the glucocorticoid receptor. Biochemical and biophysical research communications 155:801-807.

Denison MS, Fisher JM and Whitlock JP, Jr. (1988) Inducible, receptor-dependent protein-DNA interactions at a dioxin-responsive transcriptional enhancer. Proceedings of the National Academy of Sciences of the United States of America 85:2528-2532.

Denison MS, Fisher JM and Whitlock JP, Jr. (1989) Protein-DNA interactions at recognition sites for the dioxin-Ah receptor complex. The Journal of biological chemistry 264:16478-16482.

Denison MS and Nagy SR (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annual review of pharmacology and toxicology 43:309-334.

Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin M and Davis RJ (1994) JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76:1025-1037.

Dhakshinamoorthy S, Long DJ, 2nd and Jaiswal AK (2000) Antioxidant regulation of genes encoding enzymes that detoxify xenobiotics and carcinogens. Current topics in cellular regulation 36:201-216.

Diaz-Villasenor A, Burns AL, Hiriart M, Cebrian ME and Ostrosky-Wegman P (2007) Arsenic-induced alteration in the expression of genes related to type 2 diabetes mellitus. Toxicology and applied pharmacology 225:123-133.

DiDonato JA, Mercurio F and Karin M (1995) Phosphorylation of I kappa B alpha precedes but is not sufficient for its dissociation from NF-kappa B. Molecular and cellular biology 15:1302-1311.

Dinkova-Kostova AT, Holtzclaw WD and Kensler TW (2005) The role of Keap1 in cellular protective responses. Chemical research in toxicology 18:1779-1791.

Douglas AP, Savage RL and Rawlins MD (1978) Paracetamol (acetaminophen) kinetics in patients with Gilber's syndrome. European journal of clinical pharmacology 13:209-212.

Dull AB, Carlson DB, Petrulis JR and Perdew GH (2002) Characterization of the phosphorylation status of the hepatitis B virus X-associated protein 2. Archives of biochemistry and biophysics 406:209-221.

Dunn DA, Pinkert CA and Kooyman DL (2005) Foundation Review: Transgenic animals and their impact on the drug discovery industry. Drug discovery today 10:757-767.

Dutton GJ (1966) Glucuronic Acid: Free and Combined, Academic Press, New York.

Dutton GJ (1978) Developmental aspects of drug conjugation, with special reference to glucuronidation. Annual review of pharmacology and toxicology 18:17-35.

Dutton GJ (1980) Acceptor substrates of UDP-glucuronosyltransferase and their assay, in Glucuronidation of Drugs and Other Compounds (Dutton GJ ed) pp 69-78, CRC Press, Boca Raton.

Dutton GJ and Storey ID (1953) The isolation of a compound of uridine diphosphate and glucuronic acid from liver. The Biochemical journal 53:xxxvii-xxxviii.

Dutton GJ and Storey ID (1954) Uridine compounds in glucuronic acid metabolism. I. The formation of glucuronides in liver suspensions. The Biochemical journal 57:275- 283.

Eguchi R, Fujimori Y, Takeda H, Tabata C, Ohta T, Kuribayashi K, Fukuoka K and Nakano T (2011) Arsenic trioxide induces apoptosis through JNK and ERK in human mesothelioma cells. Journal of cellular physiology 226:762-768.

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K and Tuschl T (2001a) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.

Elbashir SM, Lendeckel W and Tuschl T (2001b) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes & development 15:188-200.

Elion EA, Grisafi PL and Fink GR (1990) FUS3 encodes a cdc2+/CDC28-related kinase required for the transition from mitosis into conjugation. Cell 60:649-664.

Ema M, Sogawa K, Watanabe N, Chujoh Y, Matsushita N, Gotoh O, Funae Y and Fujii-Kuriyama Y (1992) cDNA cloning and structure of mouse putative Ah receptor. Biochemical and biophysical research communications 184:246-253.

Emi Y, Ikushiro S and Iyanagi T (1996) Xenobiotic responsive element-mediated transcriptional activation in the UDP-glucuronosyltransferase family 1 gene complex. The Journal of biological chemistry 271:3952-3958.

Enomoto A, Itoh K, Nagayoshi E, Haruta J, Kimura T, O'Connor T, Harada T and Yamamoto M (2001) High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicological sciences : an official journal of the Society of Toxicology 59:169-177.

Erikson E and Maller JL (1985) A protein kinase from Xenopus eggs specific for ribosomal protein S6. Proceedings of the National Academy of Sciences of the United States of America 82:742-746.

Erikson E and Maller JL (1986) Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6. The Journal of biological chemistry 261:350-355.

Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240:889-895.

Evans RM and Hollenberg SM (1988) Cooperative and positional independent transactivation domains of the human glucocorticoid receptor. Cold Spring Harbor symposia on quantitative biology 53 Pt 2:813-818.

Fagard M, Boutet S, Morel JB, Bellini C and Vaucheret H (2000) AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proceedings of the National Academy of Sciences of the United States of America 97:11650-11654.

Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK, Talalay P and Lozniewski A (2002) Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. Proceedings of the National Academy of Sciences of the United States of America 99:7610-7615.

Falany CN, Green MD, Swain E and Tephly TR (1986) Substrate specificity and characterization of rat liver p-nitrophenol, 3 alpha-hydroxysteroid and 17 beta- hydroxysteroid UDP-glucuronosyltransferases. The Biochemical journal 238:65-73.

Falany CN and Tephly TR (1983) Separation, purification and characterization of three isoenzymes of UDP-glucuronyltransferase from rat liver microsomes. Archives of biochemistry and biophysics 227:248-258.

Favreau LV and Pickett CB (1991) Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants. The Journal of biological chemistry 266:4556-4561.

Favreau LV and Pickett CB (1993) Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Characterization of a DNA-protein interaction at the antioxidant responsive element and induction by 12-O-tetradecanoylphorbol 13- acetate. The Journal of biological chemistry 268:19875-19881.

Fawell SE, Lees JA, White R and Parker MG (1990) Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor. Cell 60:953-962.

Finco TS, Beg AA and Baldwin AS, Jr. (1994) Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors. Proceedings of the National Academy of Sciences of the United States of America 91:11884-11888.

Forman BM and Evans RM (1995) Nuclear hormone receptors activate direct, inverted, and everted repeats. Annals of the New York Academy of Sciences 761:29- 37.

Forman BM, Tzameli I, Choi HS, Chen J, Simha D, Seol W, Evans RM and Moore DD (1998) Androstane metabolites bind to and deactivate the nuclear receptor CAR- beta. Nature 395:612-615.

Francis GA, Fayard E, Picard F and Auwerx J (2003) Nuclear receptors and the control of metabolism. Annual review of physiology 65:261-311.

Fremont JJ, Wang RW and King CD (2005) Coimmunoprecipitation of UDP- glucuronosyltransferase isoforms and cytochrome P450 3A4. Molecular pharmacology 67:260-262.

Freshney NW, Rawlinson L, Guesdon F, Jones E, Cowley S, Hsuan J and Saklatvala J (1994) Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78:1039-1049.

Friling RS, Bensimon A, Tichauer Y and Daniel V (1990) Xenobiotic-inducible expression of murine glutathione S-transferase Ya subunit gene is controlled by an electrophile-responsive element. Proceedings of the National Academy of Sciences of the United States of America 87:6258-6262.

Fujisawa-Sehara A, Sogawa K, Yamane M and Fujii-Kuriyama Y (1987) Characterization of xenobiotic responsive elements upstream from the drug- metabolizing cytochrome P-450c gene: a similarity to glucocorticoid regulatory elements. Nucleic acids research 15:4179-4191.

Fujiwara R, Chen S, Karin M and Tukey RH (2012) Reduced expression of UGT1A1 in intestines of humanized UGT1 mice via inactivation of NF-kappaB leads to hyperbilirubinemia. Gastroenterology 142:109-118 e102.

Fujiwara R, Nakajima M, Oda S, Yamanaka H, Ikushiro S, Sakaki T and Yokoi T (2010a) Interactions between human UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A enzymes. Journal of pharmaceutical sciences 99:442-454.

Fujiwara R, Nguyen N, Chen S and Tukey RH (2010b) Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus. Proceedings of the National Academy of Sciences of the United States of America 107:5024-5029.

Galijatovic A, Otake Y, Walle UK and Walle T (2001) Induction of UDP- glucuronosyltransferase UGT1A1 by the flavonoid chrysin in Caco-2 cells--potential role in carcinogen bioinactivation. Pharmaceutical research 18:374-379.

Galijatovic A, Walle UK and Walle T (2000) Induction of UDP- glucuronosyltransferase by the flavonoids chrysin and quercetin in Caco-2 cells. Pharmaceutical research 17:21-26.

Ghosh S and Baltimore D (1990) Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature 344:678-682.

Gilmore T and Martin GS (1983) Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine. Nature 306:487-490.

Gilmore TD (1999) The Rel/NF-kappaB signal transduction pathway: introduction. Oncogene 18:6842-6844.

Glass CK (1994) Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. Endocrine reviews 15:391-407.

Goldstein JA, Hickman P, Bergman H, McKinney JD and Walker MP (1977) Separation of pure polychlorinated biphenyl isomers into two types of inducers on the basis of induction of cytochrome P-450 or P-448. Chemico-biological interactions 17:69-87.

Gomez N and Cohen P (1991) Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature 353:170-173.

Gong QH, Cho JW, Huang T, Potter C, Gholami N, Basu NK, Kubota S, Carvalho S, Pennington MW, Owens IS and Popescu NC (2001) Thirteen UDPglucuronosyltransferase genes are encoded at the human UGT1 gene complex locus. Pharmacogenetics 11:357-368.

Gonzalez FJ, Fernandez-Salguero P and Ward JM (1996) The role of the aryl hydrocarbon receptor in animal development, physiological homeostasis and toxicity of TCDD. The Journal of toxicological sciences 21:273-277.

Gonzalez FJ and Nebert DW (1990) Evolution of the P450 gene superfamily: animal- plant 'warfare', molecular drive and human genetic differences in drug oxidation. Trends in genetics : TIG 6:182-186.

Gordon JW and Ruddle FH (1981) Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214:1244-1246.

Gorski JP and Kasper CB (1977) Purification and properties of microsomal UDP- glucuronosyltransferase from rat liver. The Journal of biological chemistry 252:1336- 1343.

Gourdon G, Morle F, Roche J, Tourneur N, Joulain V and Godet J (1992) Identification of GATA-1 and NF-E2 binding sites in the flanking regions of the human alpha-globin genes. Acta haematologica 87:136-144.

Gourley GR (1997) Bilirubin metabolism and kernicterus. Advances in pediatrics 44:173-229.

Gram TE, Hansen AR and Fouts JR (1968) The submicrosomal distribution of hepatic uridine diphosphate glucuronyltransferases in the rabbit. The Biochemical journal 106:587-591.

Green MD, Oturu EM and Tephly TR (1994) Stable expression of a human liver UDP- glucuronosyltransferase (UGT2B15) with activity toward steroid and xenobiotic

substrates. Drug metabolism and disposition: the biological fate of chemicals 22:799- 805.

Gressner AM and Wool IG (1976) Effect of experimental diabetes and insulin on phosphorylation of rat liver ribosomal protein S6. Nature 259:148-150.

Gschaidmeier H and Bock KW (1994) Radiation inactivation analysis of microsomal UDP-glucuronosyltransferases catalysing mono- and diglucuronide formation of 3,6- dihydroxybenzo(a)pyrene and 3,6-dihydroxychrysene. Biochemical pharmacology 48:1545-1549.

Gu YZ, Hogenesch JB and Bradfield CA (2000) The PAS superfamily: sensors of environmental and developmental signals. Annual review of pharmacology and toxicology 40:519-561.

Guillemette C (2003) Pharmacogenomics of human UDP-glucuronosyltransferase enzymes. The pharmacogenomics journal 3:136-158.

Guillemette C, Belanger A and Lepine J (2004) Metabolic inactivation of estrogens in breast tissue by UDP-glucuronosyltransferase enzymes: an overview. Breast cancer research : BCR 6:246-254.

Guillemette C, Millikan RC, Newman B and Housman DE (2000a) Genetic polymorphisms in uridine diphospho-glucuronosyltransferase 1A1 and association with breast cancer among African Americans. Cancer research 60:950-956.

Guillemette C, Ritter JK, Auyeung DJ, Kessler FK and Housman DE (2000b) Structural heterogeneity at the UDP-glucuronosyltransferase 1 locus: functional consequences of three novel missense mutations in the human UGT1A7 gene. Pharmacogenetics 10:629-644.

Guo S and Kemphues KJ (1995) par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81:611-620.

Hahn ME (1998) The aryl hydrocarbon receptor: a comparative perspective. Comparative biochemistry and physiology Part C, Pharmacology, toxicology & endocrinology 121:23-53.

Hahn ME (2002) Aryl hydrocarbon receptors: diversity and evolution. Chemico- biological interactions 141:131-160.

Hamilton AJ and Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286:950-952.

Han J, Lee JD, Bibbs L and Ulevitch RJ (1994) A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265:808-811.

Handschin C, Podvinec M and Meyer UA (2000) CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Proceedings of the National Academy of Sciences of the United States of America 97:10769-10774.

Hankinson O (1995) The aryl hydrocarbon receptor complex. Annual review of pharmacology and toxicology 35:307-340.

Harding D, Jeremiah SJ, Povey S and Burchell B (1990) Chromosomal mapping of a human phenol UDP-glucuronosyltransferase, GNT1. Annals of human genetics 54:17- 21.

Harding D, Wilson SM, Jackson MR, Burchell B, Green MD and Tephly TR (1987) Nucleotide and deduced amino acid sequence of rat liver 17 beta-hydroxysteroid UDP-glucuronosyltransferase. Nucleic acids research 15:3936.

Haselbacher GK, Humbel RE and Thomas G (1979) Insulin-like growth factor: insulin or serum increase phosphorylation of ribosomal protein S6 during transition of stationary chick embryo fibroblasts into early G1 phase of the cell cycle. FEBS letters 100:185-190.

Heid SE, Pollenz RS and Swanson HI (2000) Role of heat shock protein 90 dissociation in mediating agonist-induced activation of the aryl hydrocarbon receptor. Molecular pharmacology 57:82-92.

Henkel T, Machleidt T, Alkalay I, Kronke M, Ben-Neriah Y and Baeuerle PA (1993) Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B. Nature 365:182-185.

Henkel T, Zabel U, van Zee K, Muller JM, Fanning E and Baeuerle PA (1992) Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-kappa B subunit. Cell 68:1121-1133.

Higginbotham GR, Huang A, Firestone D, Verrett J, Ress J and Campbell AD (1968) Chemical and toxicological evaluations of isolated and synthetic chloro derivatives of dibenzo-p-dioxin. Nature 220:702-703.

Hinson JA, Pohl LR, Monks TJ, Gillette JR and Guengerich FP (1980) 3- Hydroxyacetaminophen: a microsomal metabolite of acetaminophen. Evidence against an epoxide as the reactive metabolite of acetaminophen. Drug metabolism and disposition: the biological fate of chemicals 8:289-294.

Hinz M, Arslan SC and Scheidereit C (2012) It takes two to tango: IkappaBs, the multifunctional partners of NF-kappaB. Immunological reviews 246:59-76.

Hirayama A, Yoh K, Nagase S, Ueda A, Itoh K, Morito N, Hirayama K, Takahashi S, Yamamoto M and Koyama A (2003) EPR imaging of reducing activity in Nrf2 transcriptional factor-deficient mice. Free radical biology & medicine 34:1236-1242.

Hoffman EC, Reyes H, Chu FF, Sander F, Conley LH, Brooks BA and Hankinson O (1991) Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252:954-958.

Hollenberg SM, Giguere V, Segui P and Evans RM (1987) Colocalization of DNA- binding and transcriptional activation functions in the human glucocorticoid receptor. Cell 49:39-46.

Holtzclaw WD, Dinkova-Kostova AT and Talalay P (2004) Protection against electrophile and oxidative stress by induction of phase 2 genes: the quest for the elusive sensor that responds to inducers. Advances in enzyme regulation 44:335-367.

Honkakoski P, Sueyoshi T and Negishi M (2003) Drug-activated nuclear receptors CAR and PXR. Annals of medicine 35:172-182.

Honkakoski P, Zelko I, Sueyoshi T and Negishi M (1998) The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Molecular and cellular biology 18:5652-5658.

Hu Z and Wells PG (1992) In vitro and in vivo biotransformation and covalent binding of benzo(a)pyrene in Gunn and RHA rats with a genetic deficiency in bilirubin uridine diphosphate-glucuronosyltransferase. The Journal of pharmacology and experimental therapeutics 263:334-342.

Hu Z and Wells PG (1994) Modulation of benzo[a]pyrene bioactivation by glucuronidation in lymphocytes and hepatic microsomes from rats with a hereditary deficiency in bilirubin UDP-glucuronosyltransferase. Toxicology and applied pharmacology 127:306-313.

Huang W, Zhang J, Chua SS, Qatanani M, Han Y, Granata R and Moore DD (2003) Induction of bilirubin clearance by the constitutive androstane receptor (CAR). Proceedings of the National Academy of Sciences of the United States of America 100:4156-4161.

Hum DW, Belanger A, Levesque E, Barbier O, Beaulieu M, Albert C, Vallee M, Guillemette C, Tchernof A, Turgeon D and Dubois S (1999) Characterization of UDP- glucuronosyltransferases active on steroid hormones. The Journal of steroid biochemistry and molecular biology 69:413-423.

Hunter T and Cooper JA (1985) Protein-tyrosine kinases. Annual review of biochemistry 54:897-930.

Huxford T, Huang DB, Malek S and Ghosh G (1998) The crystal structure of the IkappaBalpha/NF-kappaB complex reveals mechanisms of NF-kappaB inactivation. Cell 95:759-770.

Hyun JS, Satsu H and Shimizu M (2007) Cadmium induces interleukin-8 production via NF-kappaB activation in the human intestinal epithelial cell, Caco-2. Cytokine 37:26-34.

Ikushiro S, Emi Y and Iyanagi T (1997) Protein-protein interactions between UDP- glucuronosyltransferase isozymes in rat hepatic microsomes. Biochemistry 36:7154- 7161.

Ikushiro S, Sahara M, Emi Y, Yabusaki Y and Iyanagi T (2004) Functional co- expression of xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 and UDP- glucuronosyltransferase 1A6, in yeast microsomes. Biochimica et biophysica acta 1672:86-92.

Ikuta T, Eguchi H, Tachibana T, Yoneda Y and Kawajiri K (1998) Nuclear localization and export signals of the human aryl hydrocarbon receptor. The Journal of biological chemistry 273:2895-2904.

Ikuta T and Kan YW (1991) In vivo protein-DNA interactions at the beta-globin gene locus. Proceedings of the National Academy of Sciences of the United States of America 88:10188-10192.

Ingebritsen TS, Lee HS, Parker RA and Gibson DM (1978) Reversible modulation of the activities of both liver microsomal hydroxymethylglutaryl coenzyme A reductase and its inactivating enzyme. Evidence for regulation by phosphorylation- dephosphorylation. Biochemical and biophysical research communications 81:1268- 1277.

Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, Bannai S and Yamamoto M (2000) Transcription factor Nrf2 coordinately regulates a group of oxidative stress- inducible genes in macrophages. The Journal of biological chemistry 275:16023- 16029.

Ishii Y, Iwanaga M, Nishimura Y, Takeda S, Ikushiro S, Nagata K, Yamazoe Y, Mackenzie PI and Yamada H (2007) Protein-protein interactions between rat hepatic cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs): evidence for the functionally active UGT in P450-UGT complex. Drug metabolism and pharmacokinetics 22:367-376.

Ishii Y, Takeda S and Yamada H (2010) Modulation of UDP-glucuronosyltransferase activity by protein-protein association. Drug metabolism reviews 42:145-158.

Ishii Y, Takeda S, Yamada H and Oguri K (2005) Functional protein-protein interaction of drug metabolizing enzymes. Frontiers in bioscience : a journal and virtual library 10:887-895.

Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M and Nabeshima Y (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochemical and biophysical research communications 236:313-322.

Itoh K, Igarashi K, Hayashi N, Nishizawa M and Yamamoto M (1995) Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor heterodimerizing with the small Maf family proteins. Molecular and cellular biology 15:4184-4193.

Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD and Yamamoto M (1999) Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes & development 13:76-86.

Iyanagi T, Haniu M, Sogawa K, Fujii-Kuriyama Y, Watanabe S, Shively JE and Anan KF (1986) Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase. The Journal of biological chemistry 261:15607-15614.

Iyer M, Reschly EJ and Krasowski MD (2006) Functional evolution of the pregnane X receptor. Expert opinion on drug metabolism & toxicology 2:381-397.

Jackson MR and Burchell B (1986) The full length coding sequence of rat liver androsterone UDP-glucuronyltransferase cDNA and comparison with other members of this gene family. Nucleic acids research 14:779-795.

Jackson MR, McCarthy LR, Corser RB, Barr GC and Burchell B (1985) Cloning of cDNAs coding for rat hepatic microsomal UDP-glucuronyltransferases. Gene 34:147- 153.

Jackson MR, McCarthy LR, Harding D, Wilson S, Coughtrie MW and Burchell B (1987) Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA. The Biochemical journal 242:581-588.

Jaiswal AK (2000) Regulation of genes encoding NAD(P)H:quinone oxidoreductases. Free radical biology & medicine 29:254-262.

Jansen PL, Bosma PJ and Chowdhury JR (1995) Molecular biology of bilirubin metabolism. Progress in liver diseases 13:125-150.

Jedlitschky G, Cassidy AJ, Sales M, Pratt N and Burchell B (1999) Cloning and characterization of a novel human olfactory UDP-glucuronosyltransferase. The Biochemical journal 340 ( Pt 3):837-843.

Jiang W, Xu B, Wu B, Yu R and Hu M (2012) UDP-glucuronosyltransferase (UGT) 1A9-overexpressing HeLa cells is an appropriate tool to delineate the kinetic interplay between breast cancer resistance protein (BRCP) and UGT and to rapidly identify the glucuronide substrates of BCRP. Drug metabolism and disposition: the biological fate of chemicals 40:336-345.

Jin CJ, Mackenzie PI and Miners JO (1997) The regio- and stereo-selectivity of C19 and C21 hydroxysteroid glucuronidation by UGT2B7 and UGT2B11. Archives of biochemistry and biophysics 341:207-211.

Jin CJ, Miners JO, Lillywhite KJ and Mackenzie PI (1993) cDNA cloning and expression of two new members of the human liver UDP-glucuronosyltransferase 2B subfamily. Biochemical and biophysical research communications 194:496-503.

Jones SA, Moore LB, Shenk JL, Wisely GB, Hamilton GA, McKee DD, Tomkinson NC, LeCluyse EL, Lambert MH, Willson TM, Kliewer SA and Moore JT (2000) The pregnane X receptor: a promiscuous xenobiotic receptor that has diverged during evolution. Mol Endocrinol 14:27-39.

Kadakol A, Ghosh SS, Sappal BS, Sharma G, Chowdhury JR and Chowdhury NR (2000) Genetic lesions of bilirubin uridine-diphosphoglucuronate glucuronosyltransferase (UGT1A1) causing Crigler-Najjar and Gilbert syndromes: correlation of genotype to phenotype. Human mutation 16:297-306.

Kaltreider RC, Davis AM, Lariviere JP and Hamilton JW (2001) Arsenic alters the function of the glucocorticoid receptor as a transcription factor. Environmental health perspectives 109:245-251.

Kanarek N and Ben-Neriah Y (2012) Regulation of NF-kappaB by ubiquitination and degradation of the IkappaBs. Immunological reviews 246:77-94.

Kapahi P, Takahashi T, Natoli G, Adams SR, Chen Y, Tsien RY and Karin M (2000) Inhibition of NF-kappa B activation by arsenite through reaction with a critical cysteine in the activation loop of Ikappa B kinase. The Journal of biological chemistry 275:36062-36066.

Karin M and Ben-Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annual review of immunology 18:621-663.

Karin M and Lin A (2002) NF-kappaB at the crossroads of life and death. Nature immunology 3:221-227.

Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K and Negishi M (1999) Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Molecular and cellular biology 19:6318-6322.

Kennerdell JR and Carthew RW (1998) Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95:1017- 1026.

Kensler TW, Wakabayashi N and Biswal S (2007) Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annual review of pharmacology and toxicology 47:89-116.

Keum YS, Owuor ED, Kim BR, Hu R and Kong AN (2003) Involvement of Nrf2 and JNK1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent phenethyl isothiocyanate (PEITC). Pharmaceutical research 20:1351-1356.

King CD, Rios GR, Assouline JA and Tephly TR (1999) Expression of UDP- glucuronosyltransferases (UGTs) 2B7 and 1A6 in the human brain and identification of 5-hydroxytryptamine as a substrate. Archives of biochemistry and biophysics 365:156-162.

Kliewer SA, Lehmann JM, Milburn MV and Willson TM (1999) The PPARs and PXRs: nuclear xenobiotic receptors that define novel hormone signaling pathways. Recent progress in hormone research 54:345-367; discussion 367-348.

Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, Perlmann T and Lehmann JM (1998) An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92:73-82.

Kliewer SA, Umesono K, Mangelsdorf DJ and Evans RM (1992) Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signalling. Nature 355:446-449.

Kligerman AD and Tennant AH (2007) Insights into the carcinogenic mode of action of arsenic. Toxicology and applied pharmacology 222:281-288.

Klug A and Schwabe JW (1995) Protein motifs 5. Zinc fingers. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 9:597-604.

Kohno M (1985) Diverse mitogenic agents induce rapid phosphorylation of a common set of cellular proteins at tyrosine in quiescent mammalian cells. The Journal of biological chemistry 260:1771-1779.

Kosako H, Gotoh Y, Matsuda S, Ishikawa M and Nishida E (1992) Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation. The EMBO journal 11:2903-2908.

Kumagai Y and Sumi D (2007) Arsenic: signal transduction, transcription factor, and biotransformation involved in cellular response and toxicity. Annual review of pharmacology and toxicology 47:243-262.

Kumar V and Chambon P (1988) The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145-156.

Kumar V, Green S, Staub A and Chambon P (1986) Localisation of the oestradiol- binding and putative DNA-binding domains of the human oestrogen receptor. The EMBO journal 5:2231-2236.

Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J and Woodgett JR (1994) The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156-160.

Lankisch TO, Gillman TC, Erichsen TJ, Ehmer U, Kalthoff S, Freiberg N, Munzel PA, Manns MP and Strassburg CP (2008) Aryl hydrocarbon receptor-mediated regulation of the human estrogen and bile acid UDP-glucuronosyltransferase 1A3 gene. Archives of toxicology 82:573-582.

Lankisch TO, Moebius U, Wehmeier M, Behrens G, Manns MP, Schmidt RE and Strassburg CP (2006) Gilbert's disease and atazanavir: from phenotype to UDP- glucuronosyltransferase haplotype. Hepatology 44:1324-1332.

Lau AT, Li M, Xie R, He QY and Chiu JF (2004) Opposed arsenite-induced signaling pathways promote cell proliferation or apoptosis in cultured lung cells. Carcinogenesis 25:21-28.

Lechner MC (1994) Cytochrome P450: Biochemistry, Biophysics and Molecular Biology, John Libbey Eurotext, Paris.

Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW and et al. (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739-746.

Lee JM, Calkins MJ, Chan K, Kan YW and Johnson JA (2003) Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. The Journal of biological chemistry 278:12029-12038.

Lee JM and Johnson JA (2004) An important role of Nrf2-ARE pathway in the cellular defense mechanism. Journal of biochemistry and molecular biology 37:139- 143.

Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT and Kliewer SA (1998) The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. The Journal of clinical investigation 102:1016-1023.

Leid M, Kastner P, Lyons R, Nakshatri H, Saunders M, Zacharewski T, Chen JY, Staub A, Garnier JM, Mader S and et al. (1992) Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell 68:377-395.

Levin DE and Errede B (1995) The proliferation of MAP kinase signaling pathways in yeast. Current opinion in cell biology 7:197-202.

Levine SL and Perdew GH (2001) Aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) activity is unaltered by phosphorylation of a periodicity/ARNT/single-minded (PAS)-region serine residue. Molecular pharmacology 59:557-566.

Li G, Lee LS, Li M, Tsao SW and Chiu JF (2011) Molecular changes during arsenic- induced cell transformation. Journal of cellular physiology 226:3225-3232.

Li Q, Van Antwerp D, Mercurio F, Lee KF and Verma IM (1999a) Severe liver degeneration in mice lacking the IkappaB kinase 2 gene. Science 284:321-325.

Li W, Harper PA, Tang BK and Okey AB (1998) Regulation of cytochrome P450 enzymes by aryl hydrocarbon receptor in human cells: CYP1A2 expression in the LS180 colon carcinoma cell line after treatment with 2,3,7,8-tetrachlorodibenzo-p- dioxin or 3-methylcholanthrene. Biochemical pharmacology 56:599-612.

Li Y and Jaiswal AK (1992) Regulation of human NAD(P)H:quinone oxidoreductase gene. Role of AP1 binding site contained within human antioxidant response element. The Journal of biological chemistry 267:15097-15104.

Li ZW, Chu W, Hu Y, Delhase M, Deerinck T, Ellisman M, Johnson R and Karin M (1999b) The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis. The Journal of experimental medicine 189:1839-1845.

Li-Masters T and Morgan ET (2002) Down-regulation of phenobarbital-induced cytochrome P4502B mRNAs and proteins by endotoxin in mice: independence from nitric oxide production by inducible nitric oxide synthase. Biochemical pharmacology 64:1703-1711.

Lipschitz WL and Bueding E (1939) Mechanism of the biological formation of conjugated glucuronic acids. The Journal of biological chemistry 129:333-358.

Liu X, Tam VH and Hu M (2007) Disposition of flavonoids via enteric recycling: determination of the UDP-glucuronosyltransferase isoforms responsible for the metabolism of flavonoids in intact Caco-2 TC7 cells using siRNA. Molecular pharmaceutics 4:873-882.

Liu ZG, Hsu H, Goeddel DV and Karin M (1996) Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 87:565-576.

Lohmann JU, Endl I and Bosch TC (1999) Silencing of developmental genes in Hydra. Developmental biology 214:211-214.

Luisi BF, Xu WX, Otwinowski Z, Freedman LP, Yamamoto KR and Sigler PB (1991) Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature 352:497-505.

Luo JL, Maeda S, Hsu LC, Yagita H and Karin M (2004) Inhibition of NF-kappaB in cancer cells converts inflammation- induced tumor growth mediated by TNFalpha to TRAIL-mediated tumor regression. Cancer cell 6:297-305.

Mackenzie PI (1986) Rat liver UDP-glucuronosyltransferase. Sequence and expression of a cDNA encoding a phenobarbital-inducible form. The Journal of biological chemistry 261:6119-6125.

Mackenzie PI (1987) Rat liver UDP-glucuronosyltransferase. Identification of cDNAs encoding two enzymes which glucuronidate testosterone, dihydrotestosterone, and beta-estradiol. The Journal of biological chemistry 262:9744-9749.

Mackenzie PI (1990) Expression of chimeric cDNAs in cell culture defines a region of UDP glucuronosyltransferase involved in substrate selection. The Journal of biological chemistry 265:3432-3435.

Mackenzie PI, Bock KW, Burchell B, Guillemette C, Ikushiro S, Iyanagi T, Miners JO, Owens IS and Nebert DW (2005) Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenetics and genomics 15:677- 685.

Mackenzie PI, Gonzalez FJ and Owens IS (1984a) Cell-free translation of mouse liver mRNA coding for two forms of UDP glucuronosyltransferase. Archives of biochemistry and biophysics 230:676-680.

Mackenzie PI, Gonzalez FJ and Owens IS (1984b) Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA. The Journal of biological chemistry 259:12153-12160.

Mackenzie PI, Gregory PA, Gardner-Stephen DA, Lewinsky RH, Jorgensen BR, Nishiyama T, Xie W and Radominska-Pandya A (2003) Regulation of UDP glucuronosyltransferase genes. Current drug metabolism 4:249-257.

Mackenzie PI, Hu DG and Gardner-Stephen DA (2010) The regulation of UDP- glucuronosyltransferase genes by tissue-specific and ligand-activated transcription factors. Drug metabolism reviews 42:99-109.

Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Belanger A, Fournel- Gigleux S, Green M, Hum DW, Iyanagi T, Lancet D, Louisot P, Magdalou J, Chowdhury JR, Ritter JK, Schachter H, Tephly TR, Tipton KF and Nebert DW (1997) The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 7:255-269.

Maglich JM, Sluder A, Guan X, Shi Y, McKee DD, Carrick K, Kamdar K, Willson TM and Moore JT (2001) Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes. Genome biology 2:RESEARCH0029.

Maglich JM, Stoltz CM, Goodwin B, Hawkins-Brown D, Moore JT and Kliewer SA (2002) Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification. Molecular pharmacology 62:638-646.

Makris C, Godfrey VL, Krahn-Senftleben G, Takahashi T, Roberts JL, Schwarz T, Feng L, Johnson RS and Karin M (2000) Female mice heterozygous for IKK gamma/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Molecular cell 5:969-979.

Maller JL (1986) Mitogenic signalling and protein phosphorylation in Xenopus oocytes. Journal of cyclic nucleotide and protein phosphorylation research 11:543- 555.

Maller JL, Foulkes JG, Erikson E and Baltimore D (1985) Phosphorylation of ribosomal protein S6 on serine after microinjection of the Abelson murine leukemia virus tyrosine-specific protein kinase into Xenopus oocytes. Proceedings of the National Academy of Sciences of the United States of America 82:272-276.

Mangelsdorf DJ and Evans RM (1995) The RXR heterodimers and orphan receptors. Cell 83:841-850.

Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P and Evans RM (1995) The nuclear receptor superfamily: the second decade. Cell 83:835-839.

Marks MS, Hallenbeck PL, Nagata T, Segars JH, Appella E, Nikodem VM and Ozato K (1992) H-2RIIBP (RXR beta) heterodimerization provides a mechanism for combinatorial diversity in the regulation of retinoic acid and thyroid hormone responsive genes. The EMBO journal 11:1419-1435.

Matern H, Matern S and Gerok W (1982) Isolation and characterization of rat liver microsomal UDP-glucuronosyltransferase activity toward chenodeoxycholic acid and testosterone as a single form of enzyme. The Journal of biological chemistry 257:7422-7429.

Matern S, Matern H, Farthmann EH and Gerok W (1984) Hepatic and extrahepatic glucuronidation of bile acids in man. Characterization of bile acid uridine 5'- diphosphate-glucuronosyltransferase in hepatic, renal, and intestinal microsomes. The Journal of clinical investigation 74:402-410.

Matic M, Mahns A, Tsoli M, Corradin A, Polly P and Robertson GR (2007) Pregnane X receptor: promiscuous regulator of detoxification pathways. The international journal of biochemistry & cell biology 39:478-483.

Matsui M and Nagai F (1986) Genetic deficiency of androsterone UDP- glucuronosyltransferase activity in Wistar rats is due to the loss of enzyme protein. The Biochemical journal 234:139-144.

McDonnell WM, Hitomi E and Askari FK (1996) Identification of bilirubin UDP-GTs in the human alimentary tract in accordance with the gut as a putative metabolic organ. Biochemical pharmacology 51:483-488.

McGurk KA, Brierley CH and Burchell B (1998) Drug glucuronidation by human renal UDP-glucuronosyltransferases. Biochemical pharmacology 55:1005-1012.

McMahon M, Itoh K, Yamamoto M, Chanas SA, Henderson CJ, McLellan LI, Wolf CR, Cavin C and Hayes JD (2001) The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer research 61:3299-3307.

McManus MT and Sharp PA (2002) Gene silencing in mammals by small interfering RNAs. Nature reviews Genetics 3:737-747.

Medina-Diaz IM, Estrada-Muniz E, Reyes-Hernandez OD, Ramirez P, Vega L and Elizondo G (2009) Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Toxicology and applied pharmacology 239:162-168.

Meech R and Mackenzie PI (1997) UDP-glucuronosyltransferase, the role of the amino terminus in dimerization. The Journal of biological chemistry 272:26913- 26917.

Mellits KH, Hay RT and Goodbourn S (1993) Proteolytic degradation of MAD3 (I kappa B alpha) and enhanced processing of the NF-kappa B precursor p105 are obligatory steps in the activation of NF-kappa B. Nucleic acids research 21:5059- 5066.

Melton PE, Haack K, Goring HH, Laston S, Umans JG, Lee ET, Fabsitz RR, Devereux RB, Best LG, Maccluer JW, Almasy L and Cole SA (2011) Genetic influences on serum bilirubin in American Indians: The Strong Heart Family Study. American journal of human biology : the official journal of the Human Biology Council 23:118-125.

Meyer BK and Perdew GH (1999) Characterization of the AhR-hsp90-XAP2 core complex and the role of the immunophilin-related protein XAP2 in AhR stabilization. Biochemistry 38:8907-8917.

Meyer BK, Pray-Grant MG, Vanden Heuvel JP and Perdew GH (1998) Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor core complex and exhibits transcriptional enhancer activity. Molecular and cellular biology 18:978-988.

Miller J, McLachlan AD and Klug A (1985) Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. The EMBO journal 4:1609- 1614.

Misquitta L and Paterson BM (1999) Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation. Proceedings of the National Academy of Sciences of the United States of America 96:1451-1456.

Moi P, Chan K, Asunis I, Cao A and Kan YW (1994) Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proceedings of the National Academy of Sciences of the United States of America 91:9926-9930.

Moi P and Kan YW (1990) Synergistic enhancement of globin gene expression by activator protein-1-like proteins. Proceedings of the National Academy of Sciences of the United States of America 87:9000-9004.

Monaghan G, Burchell B and Boxer M (1997) Structure of the human UGT2B4 gene encoding a bile acid UDP-glucuronosyltransferase. Mammalian genome : official journal of the International Mammalian Genome Society 8:692-694.

Monaghan G, Clarke DJ, Povey S, See CG, Boxer M and Burchell B (1994) Isolation of a human YAC contig encompassing a cluster of UGT2 genes and its regional localization to chromosome 4q13. Genomics 23:496-499.

Moore LB, Maglich JM, McKee DD, Wisely B, Willson TM, Kliewer SA, Lambert MH and Moore JT (2002) Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically distinct classes of nuclear receptors. Mol Endocrinol 16:977-986.

Mulder GJ (1971) The heterogeneity of uridine diphosphate glucuronyltransferase from rat liver. The Biochemical journal 125:9-15.

Muntane-Relat J, Ourlin JC, Domergue J and Maurel P (1995) Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. Hepatology 22:1143-1153.

Munzel PA, Lehmkoster T, Bruck M, Ritter JK and Bock KW (1998) Aryl hydrocarbon receptor-inducible or constitutive expression of human UDP glucuronosyltransferase UGT1A6. Archives of biochemistry and biophysics 350:72- 78.

Munzel PA, Schmohl S, Buckler F, Jaehrling J, Raschko FT, Kohle C and Bock KW (2003) Contribution of the Ah receptor to the phenolic antioxidant-mediated expression of human and rat UDP-glucuronosyltransferase UGT1A6 in Caco-2 and rat hepatoma 5L cells. Biochemical pharmacology 66:841-847.

Murre C, McCaw PS and Baltimore D (1989a) A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56:777-783.

Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB and et al. (1989b) Interactions between heterologous helix- loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58:537-544.

Nakamura KD, Martinez R and Weber MJ (1983) Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts. Molecular and cellular biology 3:380-390.

Nakielny S, Campbell DG and Cohen P (1992) MAP kinase kinase from rabbit skeletal muscle. A novel dual specificity enzyme showing homology to yeast protein kinases involved in pheromone-dependent signal transduction. FEBS letters 308:183- 189.

Navas-Acien A, Sharrett AR, Silbergeld EK, Schwartz BS, Nachman KE, Burke TA and Guallar E (2005) Arsenic exposure and cardiovascular disease: a systematic

review of the epidemiologic evidence. American journal of epidemiology 162:1037- 1049.

Nebert DW (1997) Polymorphisms in drug-metabolizing enzymes: what is their clinical relevance and why do they exist? American journal of human genetics 60:265- 271.

Nebert DW (2006) Drug Metabolism: Evolution, in eLS, John Wiley & Sons Ltd, Chichester.

Nebert DW and Dieter MZ (2000) The evolution of drug metabolism. Pharmacology 61:124-135.

Nellen W and Lichtenstein C (1993) What makes an mRNA anti-sense-itive? Trends in biochemical sciences 18:419-423.

Ney PA, Sorrentino BP, McDonagh KT and Nienhuis AW (1990) Tandem AP-1- binding sites within the human beta-globin dominant control region function as an inducible enhancer in erythroid cells. Genes & development 4:993-1006.

Ngo H, Tschudi C, Gull K and Ullu E (1998) Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proceedings of the National Academy of Sciences of the United States of America 95:14687-14692.

Nguyen N and Tukey RH (1997) Baculovirus-directed expression of rabbit UDP- glucuronosyltransferases in Spodoptera frugiperda cells. Drug metabolism and disposition: the biological fate of chemicals 25:745-749.

Nguyen T, Huang HC and Pickett CB (2000) Transcriptional regulation of the antioxidant response element. Activation by Nrf2 and repression by MafK. The Journal of biological chemistry 275:15466-15473.

Nguyen T, Sherratt PJ, Nioi P, Yang CS and Pickett CB (2005) Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1. The Journal of biological chemistry 280:32485-32492.

Nioi P, McMahon M, Itoh K, Yamamoto M and Hayes JD (2003) Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence. The Biochemical journal 374:337-348.

Noreault TL, Kostrubsky VE, Wood SG, Nichols RC, Strom SC, Trask HW, Wrighton SA, Evans RM, Jacobs JM, Sinclair PR and Sinclair JF (2005) Arsenite decreases CYP3A4 and RXRalpha in primary human hepatocytes. Drug metabolism and disposition: the biological fate of chemicals 33:993-1003.

Novak-Hofer I and Thomas G (1984) An activated S6 kinase in extracts from serum- and epidermal growth factor-stimulated Swiss 3T3 cells. The Journal of biological chemistry 259:5995-6000.

Ockenga J, Vogel A, Teich N, Keim V, Manns MP and Strassburg CP (2003) UDP glucuronosyltransferase (UGT1A7) gene polymorphisms increase the risk of chronic pancreatitis and pancreatic cancer. Gastroenterology 124:1802-1808.

Okey AB, Riddick DS and Harper PA (1994) The Ah receptor: mediator of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Toxicology letters 70:1-22.

Omiecinski CJ, Vanden Heuvel JP, Perdew GH and Peters JM (2011) Xenobiotic metabolism, disposition, and regulation by receptors: from biochemical phenomenon to predictors of major toxicities. Toxicological sciences : an official journal of the Society of Toxicology 120 Suppl 1:S49-75.

Operaña TN, Nguyen N, Chen S, Beaton D and Tukey RH (2007) Human CYP1A1GFP expression in transgenic mice serves as a biomarker for environmental toxicant exposure. Toxicological sciences : an official journal of the Society of Toxicology 95:98-107.

Operaña TN and Tukey RH (2007) Oligomerization of the UDP- glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation. The Journal of biological chemistry 282:4821-4829.

Ou Z, Huang M, Zhao L and Xie W (2010) Use of transgenic mice in UDP- glucuronosyltransferase (UGT) studies. Drug metabolism reviews 42:123-131.

Owens D and Evans J (1975) Population studies on Gilbert's syndrome. Journal of medical genetics 12:152-156.

Pacifici GM, Franchi M, Bencini C, Repetti F, Di Lascio N and Muraro GB (1988) Tissue distribution of drug-metabolizing enzymes in humans. Xenobiotica; the fate of foreign compounds in biological systems 18:849-856.

Pacifici GM, Giuliani L and Calcaprina R (1986) Glucuronidation of 1-naphthol in nuclear and microsomal fractions of the human intestine. Pharmacology 33:103-109.

Pahl HL (1999) Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18:6853-6866.

Palombella VJ, Rando OJ, Goldberg AL and Maniatis T (1994) The ubiquitin- proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell 78:773-785.

Parquet M, Pessah M, Sacquet E, Salvat C, Raizman A and Infante R (1985) Glucuronidation of bile acids in human liver, intestine and kidney. An in vitro study on hyodeoxycholic acid. FEBS letters 189:183-187.

Pascussi JM, Gerbal-Chaloin S, Pichard-Garcia L, Daujat M, Fabre JM, Maurel P and Vilarem MJ (2000) Interleukin-6 negatively regulates the expression of pregnane X receptor and constitutively activated receptor in primary human hepatocytes. Biochemical and biophysical research communications 274:707-713.

Pellequer JL, Wager-Smith KA, Kay SA and Getzoff ED (1998) Photoactive yellow protein: a structural prototype for the three-dimensional fold of the PAS domain superfamily. Proceedings of the National Academy of Sciences of the United States of America 95:5884-5890.

Perdew GH (1988) Association of the Ah receptor with the 90-kDa heat shock protein. The Journal of biological chemistry 263:13802-13805.

Peters WH, Allebes WA, Jansen PL, Poels LG and Capel PJ (1987) Characterization and tissue specificity of a monoclonal antibody against human uridine 5'-diphosphate- glucuronosyltransferase. Gastroenterology 93:162-169.

Peters WH and Jansen PL (1988) Immunocharacterization of UDP- glucuronyltransferase isoenzymes in human liver, intestine and kidney. Biochemical pharmacology 37:564-567.

Peters WH, Jansen PL and Nauta H (1984) The molecular weights of UDP- glucuronyltransferase determined with radiation-inactivation analysis. A molecular model of bilirubin UDP-glucuronyltransferase. The Journal of biological chemistry 259:11701-11705.

Peters WH, Kock L, Nagengast FM and Kremers PG (1991) Biotransformation enzymes in human intestine: critical low levels in the colon? Gut 32:408-412.

Peters WH, Nagengast FM and van Tongeren JH (1989) Glutathione S-transferase, cytochrome P450, and uridine 5'-diphosphate-glucuronosyltransferase in human small intestine and liver. Gastroenterology 96:783-789.

Pi J, Qu W, Reece JM, Kumagai Y and Waalkes MP (2003) Transcription factor Nrf2 activation by inorganic arsenic in cultured keratinocytes: involvement of hydrogen peroxide. Experimental cell research 290:234-245.

Poland A and Glover E (1973a) 2,3,7,8-Tetrachlorodibenzo-p-dioxin: a potent inducer of -aminolevulinic acid synthetase. Science 179:476-477.

Poland A and Glover E (1973b) Chlorinated dibenzo-p-dioxins: potent inducers of delta-aminolevulinic acid synthetase and aryl hydrocarbon hydroxylase. II. A study of the structure-activity relationship. Molecular pharmacology 9:736-747.

Poland A and Glover E (1974) Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. Molecular pharmacology 10:349-359.

Poland A and Glover E (1977) Chlorinated biphenyl induction of aryl hydrocarbon hydroxylase activity: a study of the structure-activity relationship. Molecular pharmacology 13:924-938.

Poland A, Glover E, Ebetino FH and Kende AS (1986) Photoaffinity labeling of the Ah receptor. The Journal of biological chemistry 261:6352-6365.

Poland A, Glover E and Kende AS (1976) Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase. The Journal of biological chemistry 251:4936-4946.

Poland A and Knutson JC (1982) 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annual review of pharmacology and toxicology 22:517-554.

Poland AP, Glover E, Robinson JR and Nebert DW (1974) Genetic expression of aryl hydrocarbon hydroxylase activity. Induction of monooxygenase activities and cytochrome P1-450 formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice genetically "nonresponsive" to other aromatic hydrocarbons. The Journal of biological chemistry 249:5599-5606.

Pollenz RS, Sattler CA and Poland A (1994) The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy. Molecular pharmacology 45:428-438.

Pryde J and Williams RT (1933) The Biochemistry and Physiology of Glucuronic Acid: The Methylation of Glucurone of Animal Origin. The Biochemical journal:1205-1209.

Qatanani M and Moore DD (2005) CAR, the continuously advancing receptor, in drug metabolism and disease. Current drug metabolism 6:329-339.

Qatanani M, Zhang J and Moore DD (2005) Role of the constitutive androstane receptor in xenobiotic-induced thyroid hormone metabolism. Endocrinology 146:995- 1002.

Radominska-Pandya A, Bratton S and Little JM (2005) A historical overview of the heterologous expression of mammalian UDP-glucuronosyltransferase isoforms over the past twenty years. Current drug metabolism 6:141-160.

Radominska-Pandya A, Czernik PJ, Little JM, Battaglia E and Mackenzie PI (1999) Structural and functional studies of UDP-glucuronosyltransferases. Drug metabolism reviews 31:817-899.

Radu P and Atsmon J (2001) Gilbert's syndrome--clinical and pharmacological implications. The Israel Medical Association journal : IMAJ 3:593-598.

Ramos-Gomez M, Kwak MK, Dolan PM, Itoh K, Yamamoto M, Talalay P and Kensler TW (2001) Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 98:3410-3415.

Rao DD, Vorhies JS, Senzer N and Nemunaitis J (2009) siRNA vs. shRNA: similarities and differences. Advanced drug delivery reviews 61:746-759.

Rao M and Sockanathan S (2005) Molecular mechanisms of RNAi: implications for development and disease. Birth defects research Part C, Embryo today : reviews 75:28-42.

Ray LB and Sturgill TW (1988a) Insulin-stimulated microtubule associated protein kinase is detectable by analytical gel chromatography as a 35-kDa protein in myocytes, adipocytes, and hepatocytes. Archives of biochemistry and biophysics 262:307-313.

Ray LB and Sturgill TW (1988b) Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo. Proceedings of the National Academy of Sciences of the United States of America 85:3753-3757.

Remmel RP and Burchell B (1993) Validation and use of cloned, expressed human drug-metabolizing enzymes in heterologous cells for analysis of drug metabolism and drug-drug interactions. Biochemical pharmacology 46:559-566.

Remmel RP, Zhou J and Argikar U (2009) UDP-Glucuronosyltransferases, in Handbook of Drug Metabolism (Drugs and the Pharmaceutical Sciences) (Pearson PG and Wienkers LC eds), Informa Healthcare, New York, NY.

Renton KW and Knickle LC (1990) Regulation of hepatic cytochrome P-450 during infectious disease. Canadian journal of physiology and pharmacology 68:777-781.

Reyes H, Reisz-Porszasz S and Hankinson O (1992) Identification of the Ah receptor nuclear translocator protein (Arnt) as a component of the DNA binding form of the Ah receptor. Science 256:1193-1195.

Reynolds A, Anderson EM, Vermeulen A, Fedorov Y, Robinson K, Leake D, Karpilow J, Marshall WS and Khvorova A (2006) Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA 12:988-993.

Ritter JK, Chen F, Sheen YY, Tran HM, Kimura S, Yeatman MT and Owens IS (1992) A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini. The Journal of biological chemistry 267:3257-3261.

Ritter JK, Crawford JM and Owens IS (1991) Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells. The Journal of biological chemistry 266:1043-1047.

Ritter JK, Kessler FK, Thompson MT, Grove AD, Auyeung DJ and Fisher RA (1999) Expression and inducibility of the human bilirubin UDP-glucuronosyltransferase UGT1A1 in liver and cultured primary hepatocytes: evidence for both genetic and environmental influences. Hepatology 30:476-484.

Robinson-Rechavi M, Carpentier AS, Duffraisse M and Laudet V (2001) How many nuclear hormone receptors are there in the human genome? Trends in genetics : TIG 17:554-556.

Rosenfeld JM, Vargas R, Jr., Xie W and Evans RM (2003) Genetic profiling defines the xenobiotic gene network controlled by the nuclear receptor pregnane X receptor. Mol Endocrinol 17:1268-1282.

Rossomando AJ, Payne DM, Weber MJ and Sturgill TW (1989) Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. Proceedings of the National Academy of Sciences of the United States of America 86:6940-6943.

Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, Hunt T and Nebreda AR (1994) A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78:1027-1037.

Roussel RR and Barchowsky A (2000) Arsenic inhibits NF-kappaB-mediated gene transcription by blocking IkappaB kinase activity and IkappaBalpha phosphorylation and degradation. Archives of biochemistry and biophysics 377:204-212.

Roy Chowdhury J, Roy Chowdhury N, Falany CN, Tephly TR and Arias IM (1986) Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase. The Biochemical journal 233:827-837.

Rusconi S and Yamamoto KR (1987) Functional dissection of the hormone and DNA binding activities of the glucocorticoid receptor. The EMBO journal 6:1309-1315.

Rushmore TH, King RG, Paulson KE and Pickett CB (1990) Regulation of glutathione S-transferase Ya subunit gene expression: identification of a unique xenobiotic- responsive element controlling inducible expression by planar aromatic compounds.

100

Proceedings of the National Academy of Sciences of the United States of America 87:3826-3830.

Rushmore TH and Kong AN (2002) Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes. Current drug metabolism 3:481-490.

Rushmore TH, Morton MR and Pickett CB (1991) The antioxidant responsive element. Activation by oxidative stress and identification of the DNA consensus sequence required for functional activity. The Journal of biological chemistry 266:11632-11639.

Rushmore TH and Pickett CB (1990) Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants. The Journal of biological chemistry 265:14648-14653.

Sanchez Alvarado A and Newmark PA (1999) Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proceedings of the National Academy of Sciences of the United States of America 96:5049-5054.

Savas U, Wester MR, Griffin KJ and Johnson EF (2000) Rabbit pregnane X receptor is activated by rifampicin. Drug metabolism and disposition: the biological fate of chemicals 28:529-537.

Schaeffer HJ and Weber MJ (1999) Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Molecular and cellular biology 19:2435-2444.

Schmid R (1956) Direct-reacting bilirubin, bilirubin glucuronide, in serum, bile and urine. Science 124:76-77.

Schmidt JV and Bradfield CA (1996) Ah receptor signaling pathways. Annual review of cell and developmental biology 12:55-89.

Schmiedeberg O and Meyer H (1879) Z Physiol Chem 3.

Schmuck EM, Board PG, Whitbread AK, Tetlow N, Cavanaugh JA, Blackburn AC and Masoumi A (2005) Characterization of the monomethylarsonate reductase and dehydroascorbate reductase activities of Omega class glutathione transferase variants: implications for arsenic metabolism and the age-at-onset of Alzheimer's and Parkinson's diseases. Pharmacogenetics and genomics 15:493-501.

Schwabe JW, Chapman L, Finch JT and Rhodes D (1993) The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75:567-578.

101

Schwetz BA, Norris JM, Sparschu GL, Rowe UK, Gehring PJ, Emerson JL and Gerbig CG (1973) Toxicology of chlorinated dibenzo-p-dioxins. Environmental health perspectives 5:87-99.

Seger R, Seger D, Lozeman FJ, Ahn NG, Graves LM, Campbell JS, Ericsson L, Harrylock M, Jensen AM and Krebs EG (1992) Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases. The Journal of biological chemistry 267:25628-25631.

Sen R and Baltimore D (1986) Inducibility of kappa immunoglobulin enhancer- binding protein Nf-kappa B by a posttranslational mechanism. Cell 47:921-928.

Sen R and Baltimore D (2006) Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 1986. 46: 705-716. J Immunol 177:7485- 7496.

Senekeo-Effenberger K, Chen S, Brace-Sinnokrak E, Bonzo JA, Yueh MF, Argikar U, Kaeding J, Trottier J, Remmel RP, Ritter JK, Barbier O and Tukey RH (2007) Expression of the human UGT1 locus in transgenic mice by 4-chloro-6-(2,3-xylidino)- 2-pyrimidinylthioacetic acid (WY-14643) and implications on drug metabolism through peroxisome proliferator-activated receptor alpha activation. Drug metabolism and disposition: the biological fate of chemicals 35:419-427.

Seppen J, Bosma PJ, Goldhoorn BG, Bakker CT, Chowdhury JR, Chowdhury NR, Jansen PL and Oude Elferink RP (1994) Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase. The Journal of clinical investigation 94:2385-2391.

Sewer MB and Morgan ET (1997) Nitric oxide-independent suppression of P450 2C11 expression by interleukin-1beta and endotoxin in primary rat hepatocytes. Biochemical pharmacology 54:729-737.

Seydoux G and Fire A (1994) Soma-germline asymmetry in the distributions of embryonic RNAs in Caenorhabditis elegans. Development 120:2823-2834.

Shelby MK and Klaassen CD (2006) Induction of rat UDP-glucuronosyltransferases in liver and duodenum by microsomal enzyme inducers that activate various transcriptional pathways. Drug metabolism and disposition: the biological fate of chemicals 34:1772-1778.

Shen G, Hebbar V, Nair S, Xu C, Li W, Lin W, Keum YS, Han J, Gallo MA and Kong AN (2004) Regulation of Nrf2 transactivation domain activity. The differential effects of mitogen-activated protein kinase cascades and synergistic stimulatory effect of Raf and CREB-binding protein. The Journal of biological chemistry 279:23052- 23060.

102

Shen G and Kong AN (2009) Nrf2 plays an important role in coordinated regulation of Phase II drug metabolism enzymes and Phase III drug transporters. Biopharmaceutics & drug disposition 30:345-355.

Sieg A, Arab L, Schlierf G, Stiehl A and Kommerell B (1987) [Prevalence of Gilbert's syndrome in Germany]. Dtsch Med Wochenschr 112:1206-1208.

Simons SS, Jr., Pumphrey JG, Rudikoff S and Eisen HJ (1987) Identification of cysteine 656 as the amino acid of hepatoma tissue culture cell glucocorticoid receptors that is covalently labeled by dexamethasone 21-mesylate. The Journal of biological chemistry 262:9676-9680.

Sistonen J, Fuselli S, Palo JU, Chauhan N, Padh H and Sajantila A (2009) Pharmacogenetic variation at CYP2C9, CYP2C19, and CYP2D6 at global and microgeographic scales. Pharmacogenetics and genomics 19:170-179.

Smith CJ, Wejksnora PJ, Warner JR, Rubin CS and Rosen OM (1979) Insulin- stimulated protein phosphorylation in 3T3-L1 preadipocytes. Proceedings of the National Academy of Sciences of the United States of America 76:2725-2729.

Smith EE and Mills GT (1954) Uridine nucleotide compounds of liver. Biochimica et biophysica acta 13:386-400.

Somers DE, Schultz TF, Milnamow M and Kay SA (2000) ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis. Cell 101:319-329.

Spivack JG, Erikson RL and Maller JL (1984) Microinjection of pp60v-src into Xenopus oocytes increases phosphorylation of ribosomal protein S6 and accelerates the rate of progesterone-induced meiotic maturation. Molecular and cellular biology 4:1631-1634.

Squires EJ, Sueyoshi T and Negishi M (2004) Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver. The Journal of biological chemistry 279:49307-49314.

Srivastava DK and Bernhard SA (1987) Mechanism of transfer of reduced nicotinamide adenine dinucleotide among dehydrogenases. Transfer rates and equilibria with enzyme-enzyme complexes. Biochemistry 26:1240-1246.

Stark GR, Kerr IM, Williams BR, Silverman RH and Schreiber RD (1998) How cells respond to interferons. Annual review of biochemistry 67:227-264.

Stefanovic D, Erikson E, Pike LJ and Maller JL (1986) Activation of a ribosomal protein S6 protein kinase in Xenopus oocytes by insulin and insulin-receptor kinase. The EMBO journal 5:157-160.

103

Storey ID (1965) Some Differences in the Conjugation of O-Aminophenol and P- Nitrophenol by the Uridine Diphosphate Transglucuronylase of Mouse-Liver Homogenates. The Biochemical journal 95:209-214.

Storey ID and Dutton GJ (1955) Uridine compounds in glucuronic acid metabolism. 2. The isolation and structure of 'uridine-diphosphate-glucuronic acid'. The Biochemical journal 59:279-288.

Strassburg CP (2008) Pharmacogenetics of Gilbert's syndrome. Pharmacogenomics 9:703-715.

Strassburg CP, Manns MP and Tukey RH (1997a) Differential down-regulation of the UDP-glucuronosyltransferase 1A locus is an early event in human liver and biliary cancer. Cancer research 57:2979-2985.

Strassburg CP, Manns MP and Tukey RH (1998) Expression of the UDP- glucuronosyltransferase 1A locus in human colon. Identification and characterization of the novel extrahepatic UGT1A8. The Journal of biological chemistry 273:8719- 8726.

Strassburg CP, Nguyen N, Manns MP and Tukey RH (1999a) UDP- glucuronosyltransferase activity in human liver and colon. Gastroenterology 116:149- 160.

Strassburg CP, Obermayer-Straub P, Alex B, Durazzo M, Rizzetto M, Tukey RH and Manns MP (1996) Autoantibodies against glucuronosyltransferases differ between viral hepatitis and autoimmune hepatitis. Gastroenterology 111:1576-1586.

Strassburg CP, Oldhafer K, Manns MP and Tukey RH (1997b) Differential expression of the UGT1A locus in human liver, biliary, and gastric tissue: identification of UGT1A7 and UGT1A10 transcripts in extrahepatic tissue. Molecular pharmacology 52:212-220.

Strassburg CP, Strassburg A, Nguyen N, Li Q, Manns MP and Tukey RH (1999b) Regulation and function of family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus. The Biochemical journal 338 ( Pt 2):489- 498.

Strassburg CP, Vogel A, Kneip S, Tukey RH and Manns MP (2002) Polymorphisms of the human UDP-glucuronosyltransferase (UGT) 1A7 gene in colorectal cancer. Gut 50:851-856.

Sturgill TW and Ray LB (1986) Muscle proteins related to microtubule associated protein-2 are substrates for an insulin-stimulatable kinase. Biochemical and biophysical research communications 134:565-571.

104

Sturgill TW, Ray LB, Erikson E and Maller JL (1988) Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 334:715- 718.

Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P and Negishi M (1999) The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. The Journal of biological chemistry 274:6043-6046.

Sueyoshi T and Negishi M (2001) Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annual review of pharmacology and toxicology 41:123-143.

Sugatani J, Kojima H, Ueda A, Kakizaki S, Yoshinari K, Gong QH, Owens IS, Negishi M and Sueyoshi T (2001) The phenobarbital response enhancer module in the human bilirubin UDP-glucuronosyltransferase UGT1A1 gene and regulation by the nuclear receptor CAR. Hepatology 33:1232-1238.

Sugatani J, Mizushima K, Osabe M, Yamakawa K, Kakizaki S, Takagi H, Mori M, Ikari A and Miwa M (2008) Transcriptional regulation of human UGT1A1 gene expression through distal and proximal promoter motifs: implication of defects in the UGT1A1 gene promoter. Naunyn-Schmiedeberg's archives of pharmacology 377:597- 605.

Sugatani J, Nishitani S, Yamakawa K, Yoshinari K, Sueyoshi T, Negishi M and Miwa M (2005) Transcriptional regulation of human UGT1A1 gene expression: activated glucocorticoid receptor enhances constitutive androstane receptor/pregnane X receptor-mediated UDP-glucuronosyltransferase 1A1 regulation with glucocorticoid receptor-interacting protein 1. Molecular pharmacology 67:845-855.

Sugatani J, Yamakawa K, Tonda E, Nishitani S, Yoshinari K, Degawa M, Abe I, Noguchi H and Miwa M (2004) The induction of human UDP-glucuronosyltransferase 1A1 mediated through a distal enhancer module by flavonoids and xenobiotics. Biochemical pharmacology 67:989-1000.

Sugatani J, Yamakawa K, Yoshinari K, Machida T, Takagi H, Mori M, Kakizaki S, Sueyoshi T, Negishi M and Miwa M (2002) Identification of a defect in the UGT1A1 gene promoter and its association with hyperbilirubinemia. Biochemical and biophysical research communications 292:492-497.

Suleman FG, Abid A, Gradinaru D, Daval JL, Magdalou J and Minn A (1998) Identification of the uridine diphosphate glucuronosyltransferase isoform UGT1A6 in rat brain and in primary cultures of neurons and astrocytes. Archives of biochemistry and biophysics 358:63-67.

105

Sun SC and Ballard DW (1999) Persistent activation of NF-kappaB by the tax transforming protein of HTLV-1: hijacking cellular IkappaB kinases. Oncogene 18:6948-6958.

Sun SC, Ganchi PA, Ballard DW and Greene WC (1993) NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science 259:1912-1915.

Svehlikova V, Wang S, Jakubikova J, Williamson G, Mithen R and Bao Y (2004) Interactions between sulforaphane and apigenin in the induction of UGT1A1 and GSTA1 in CaCo-2 cells. Carcinogenesis 25:1629-1637.

Svoboda P, Stein P, Hayashi H and Schultz RM (2000) Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development 127:4147- 4156.

Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, Fire A and Mello CC (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99:123-132.

Takeda S, Ishii Y, Iwanaga M, Mackenzie PI, Nagata K, Yamazoe Y, Oguri K and Yamada H (2005) Modulation of UDP-glucuronosyltransferase function by cytochrome P450: evidence for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4. Molecular pharmacology 67:665-672.

Talafant E (1956) Properties and composition of the bile pigment giving a direct diazo reaction. Nature 178:312.

Taura KI, Yamada H, Hagino Y, Ishii Y, Mori MA and Oguri K (2000) Interaction between cytochrome P450 and other drug-metabolizing enzymes: evidence for an association of CYP1A1 with microsomal epoxide hydrolase and UDP- glucuronosyltransferase. Biochemical and biophysical research communications 273:1048-1052.

Taylor BL and Zhulin IB (1999) PAS domains: internal sensors of oxygen, redox potential, and light. Microbiology and molecular biology reviews : MMBR 63:479- 506.

Temple AR, Clement MS and Done AK (1968) Studies of glucuronidation. IV. Evidences of different processes for o-aminophenol and p-nitrophenol. Proc Soc Exp Biol Med 128:307-314.

Tenhunen R, Marver HS and Schmid R (1969) Microsomal heme oxygenase. Characterization of the enzyme. The Journal of biological chemistry 244:6388-6394.

Tephly TR and Burchell B (1990) UDP-glucuronosyltransferases: a family of detoxifying enzymes. Trends in pharmacological sciences 11:276-279.

106

Thibaudeau J, Lepine J, Tojcic J, Duguay Y, Pelletier G, Plante M, Brisson J, Tetu B, Jacob S, Perusse L, Belanger A and Guillemette C (2006) Characterization of common UGT1A8, UGT1A9, and UGT2B7 variants with different capacities to inactivate mutagenic 4-hydroxylated metabolites of estradiol and estrone. Cancer research 66:125-133.

Thimmulappa RK, Mai KH, Srisuma S, Kensler TW, Yamamoto M and Biswal S (2002) Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. Cancer research 62:5196-5203.

Tolson AH and Wang H (2010) Regulation of drug-metabolizing enzymes by xenobiotic receptors: PXR and CAR. Advanced drug delivery reviews 62:1238-1249.

Tsai SY, Carlstedt-Duke J, Weigel NL, Dahlman K, Gustafsson JA, Tsai MJ and O'Malley BW (1988) Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation. Cell 55:361-369.

Tukey R and Tephly T (1980) Phospholipid dependency of purified estrone and p- nitrophenol UDP-glucuronyltransferases. Life sciences 27:2471-2476.

Tukey RH, Billings RE and Tephly TR (1978) Separation of oestrone UDP- glucuronyltransferase and p-nitrophenol UDP-glucuronyltransferase activities. The Biochemical journal 171:659-663.

Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annual review of pharmacology and toxicology 40:581-616.

Tukey RH and Strassburg CP (2001) Genetic multiplicity of the human UDP- glucuronosyltransferases and regulation in the gastrointestinal tract. Molecular pharmacology 59:405-414.

Tzameli I, Pissios P, Schuetz EG and Moore DD (2000) The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR. Molecular and cellular biology 20:2951-2958.

Ueda A, Hamadeh HK, Webb HK, Yamamoto Y, Sueyoshi T, Afshari CA, Lehmann JM and Negishi M (2002) Diverse roles of the nuclear orphan receptor CAR in regulating hepatic genes in response to phenobarbital. Molecular pharmacology 61:1- 6.

Uetrecht J (2010) Adverse Drug Reactions, Springer Berlin Heidelberg, Berlin, Germany.

Usui T, Kuno T and Mizutani T (2006) Induction of human UDP- glucuronosyltransferase 1A1 by cortisol-GR. Molecular biology reports 33:91-96.

107

Vahidnia A, van der Voet GB and de Wolff FA (2007) Arsenic neurotoxicity--a review. Human & experimental toxicology 26:823-832.

Vandenbrink BM, Davis JA, Pearson JT, Foti RS, Wienkers LC and Rock DA (2012) Cytochrome P450 Architecture and Cysteine Nucleophile Placement Impacts Raloxifene Mediated Mechanism-Based Inactivation. Molecular pharmacology.

Vaucheret H, Beclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Mourrain P, Palauqui JC and Vernhettes S (1998) Transgene-induced gene silencing in plants. The Plant journal : for cell and molecular biology 16:651-659.

Venugopal R and Jaiswal AK (1996) Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proceedings of the National Academy of Sciences of the United States of America 93:14960-14965.

Venugopal R and Jaiswal AK (1998) Nrf2 and Nrf1 in association with Jun proteins regulate antioxidant response element-mediated expression and coordinated induction of genes encoding detoxifying enzymes. Oncogene 17:3145-3156.

Vogel A, Kneip S, Barut A, Ehmer U, Tukey RH, Manns MP and Strassburg CP (2001) Genetic link of hepatocellular carcinoma with polymorphisms of the UDP- glucuronosyltransferase UGT1A7 gene. Gastroenterology 121:1136-1144.

Vogel A, Ockenga J, Ehmer U, Barut A, Kramer FJ, Tukey RH, Manns MP and Strassburg CP (2002) Polymorphisms of the carcinogen detoxifying UDP- glucuronosyltransferase UGT1A7 in proximal digestive tract cancer. Zeitschrift fur Gastroenterologie 40:497-502.

Vyhlidal CA, Rogan PK and Leeder JS (2004) Development and refinement of pregnane X receptor (PXR) DNA binding site model using information theory: insights into PXR-mediated gene regulation. The Journal of biological chemistry 279:46779-46786.

Wakabayashi N, Itoh K, Wakabayashi J, Motohashi H, Noda S, Takahashi S, Imakado S, Kotsuji T, Otsuka F, Roop DR, Harada T, Engel JD and Yamamoto M (2003) Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation. Nature genetics 35:238-245.

Walle T, Otake Y, Galijatovic A, Ritter JK and Walle UK (2000) Induction of UDP- glucuronosyltransferase UGT1A1 by the flavonoid chrysin in the human hepatoma cell line hep G2. Drug metabolism and disposition: the biological fate of chemicals 28:1077-1082.

108

Walsh DA, Perkins JP and Krebs EG (1968) An adenosine 3',5'-monophosphate- dependant protein kinase from rabbit skeletal muscle. The Journal of biological chemistry 243:3763-3765.

Wang CY, Mayo MW, Korneluk RG, Goeddel DV and Baldwin AS, Jr. (1998) NF- kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science 281:1680-1683.

Wang H and LeCluyse EL (2003) Role of orphan nuclear receptors in the regulation of drug-metabolising enzymes. Clinical pharmacokinetics 42:1331-1357.

Wargelius A, Ellingsen S and Fjose A (1999) Double-stranded RNA induces specific developmental defects in zebrafish embryos. Biochemical and biophysical research communications 263:156-161.

Wasserman E, Myara A, Lokiec F, Goldwasser F, Trivin F, Mahjoubi M, Misset JL and Cvitkovic E (1997) Severe CPT-11 toxicity in patients with Gilbert's syndrome: two case reports. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 8:1049-1051.

Waterhouse PM, Graham MW and Wang MB (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences of the United States of America 95:13959-13964.

Waterhouse PM, Smith NA and Wang MB (1999) Virus resistance and gene silencing: killing the messenger. Trends in plant science 4:452-457.

Weatherill PJ and Burchell B (1980) The separation and purification of rat liver UDP- glucuronyltransferase activities towards testosterone and oestrone. The Biochemical journal 189:377-380.

Weatherman RV, Fletterick RJ and Scanlan TS (1999) Nuclear-receptor ligands and ligand-binding domains. Annual review of biochemistry 68:559-581.

Wei P, Zhang J, Egan-Hafley M, Liang S and Moore DD (2000) The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nature 407:920-923.

Wells PG, Mackenzie PI, Chowdhury JR, Guillemette C, Gregory PA, Ishii Y, Hansen AJ, Kessler FK, Kim PM, Chowdhury NR and Ritter JK (2004) Glucuronidation and the UDP-glucuronosyltransferases in health and disease. Drug metabolism and disposition: the biological fate of chemicals 32:281-290.

Whitlock JP, Jr. (1999) Induction of cytochrome P4501A1. Annual review of pharmacology and toxicology 39:103-125.

109

Wianny F and Zernicka-Goetz M (2000) Specific interference with gene function by double-stranded RNA in early mouse development. Nature cell biology 2:70-75.

Williams RT (1949) Detoxication Mechanisms: The Metabolism of Drugs and Allied Organic Compounds, Chapman and Hall, London.

Williams RT (1971) The metabolism of certain drugs and food chemicals in man. Annals of the New York Academy of Sciences 179:141-154.

Willson TM, Jones SA, Moore JT and Kliewer SA (2001) Chemical genomics: functional analysis of orphan nuclear receptors in the regulation of bile acid metabolism. Medicinal research reviews 21:513-522.

Willson TM and Kliewer SA (2002) PXR, CAR and drug metabolism. Nature reviews Drug discovery 1:259-266.

Wooster R, Ebner T, Sutherland L, Clarke D and Burchell B (1993) Drug and xenobiotic glucuronidation catalysed by cloned human liver UDP- Glucuronosyltransferases stably expressed in tissue culture cell lines. Toxicology 82:119-129.

Wooster R, Sutherland L, Ebner T, Clarke D, Da Cruz e Silva O and Burchell B (1991) Cloning and stable expression of a new member of the human liver phenol/bilirubin: UDP-glucuronosyltransferase cDNA family. The Biochemical journal 278 ( Pt 2):465-469.

Wrange O and Gustafsson JA (1978) Separation of the hormone- and DNA-binding sites of the hepatic glucocorticoid receptor by means of proteolysis. The Journal of biological chemistry 253:856-865.

Wrange O, Okret S, Radojcic M, Carlstedt-Duke J and Gustafsson JA (1984) Characterization of the purified activated glucocorticoid receptor from rat liver cytosol. The Journal of biological chemistry 259:4534-4541.

Wu J, Michel H, Rossomando A, Haystead T, Shabanowitz J, Hunt DF and Sturgill TW (1992) Renaturation and partial peptide sequencing of mitogen-activated protein kinase (MAP kinase) activator from rabbit skeletal muscle. The Biochemical journal 285 ( Pt 3):701-705.

Xie T, Belinsky M, Xu Y and Jaiswal AK (1995) ARE- and TRE-mediated regulation of gene expression. Response to xenobiotics and antioxidants. The Journal of biological chemistry 270:6894-6900.

Xie W, Barwick JL, Downes M, Blumberg B, Simon CM, Nelson MC, Neuschwander-Tetri BA, Brunt EM, Guzelian PS and Evans RM (2000a) Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406:435-439.

110

Xie W, Barwick JL, Simon CM, Pierce AM, Safe S, Blumberg B, Guzelian PS and Evans RM (2000b) Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR. Genes & development 14:3014-3023.

Xie W, Radominska-Pandya A, Shi Y, Simon CM, Nelson MC, Ong ES, Waxman DJ and Evans RM (2001) An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids. Proceedings of the National Academy of Sciences of the United States of America 98:3375-3380.

Xie W, Yeuh MF, Radominska-Pandya A, Saini SP, Negishi Y, Bottroff BS, Cabrera GY, Tukey RH and Evans RM (2003) Control of steroid, heme, and carcinogen metabolism by nuclear pregnane X receptor and constitutive androstane receptor. Proceedings of the National Academy of Sciences of the United States of America 100:4150-4155.

Xu C, Li CY and Kong AN (2005) Induction of phase I, II and III drug metabolism/transport by xenobiotics. Archives of pharmacal research 28:249-268.

Yaffe SJ, Levy G, Matsuzawa T and Baliah T (1966) Enhancement of glucuronide- conjugating capacity in a hyperbilirubinemic infant due to apparent enzyme induction by phenobarbital. The New England journal of medicine 275:1461-1466.

Yancy SL, Shelden EA, Gilmont RR and Welsh MJ (2005) Sodium arsenite exposure alters cell migration, focal adhesion localization and decreases tyrosine phosphorylation of focal adhesion kinase in H9C2 myoblasts. Toxicological sciences : an official journal of the Society of Toxicology 84:278-286.

Yeh SY (1975) Urinary excretion of morphine and its metabolites in morphine- dependent subjects. The Journal of pharmacology and experimental therapeutics 192:201-210.

Yu R, Chen C, Mo YY, Hebbar V, Owuor ED, Tan TH and Kong AN (2000) Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism. The Journal of biological chemistry 275:39907-39913.

Yu R, Lei W, Mandlekar S, Weber MJ, Der CJ, Wu J and Kong AN (1999) Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals. The Journal of biological chemistry 274:27545-27552.

Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV, Naar AM, Kim SY, Boutin JM, Glass CK and Rosenfeld MG (1991) RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell 67:1251-1266.

111

Yueh MF, Huang YH, Hiller A, Chen S, Nguyen N and Tukey RH (2003) Involvement of the xenobiotic response element (XRE) in Ah receptor-mediated induction of human UDP-glucuronosyltransferase 1A1. The Journal of biological chemistry 278:15001-15006.

Yueh MF and Tukey RH (2007) Nrf2-Keap1 signaling pathway regulates human UGT1A1 expression in vitro and in transgenic UGT1 mice. The Journal of biological chemistry 282:8749-8758.

Zamore PD, Tuschl T, Sharp PA and Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25-33.

Zelko I and Negishi M (2000) Phenobarbital-elicited activation of nuclear receptor CAR in induction of cytochrome P450 genes. Biochemical and biophysical research communications 277:1-6.

Zelko I, Sueyoshi T, Kawamoto T, Moore R and Negishi M (2001) The peptide near the C terminus regulates receptor CAR nuclear translocation induced by xenochemicals in mouse liver. Molecular and cellular biology 21:2838-2846.

Zhang XK, Hoffmann B, Tran PB, Graupner G and Pfahl M (1992) Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature 355:441-446.

Zheng Z, Fang JL and Lazarus P (2002) Glucuronidation: an important mechanism for detoxification of benzo[a]pyrene metabolites in aerodigestive tract tissues. Drug metabolism and disposition: the biological fate of chemicals 30:397-403.

Zheng Z, Park JY, Guillemette C, Schantz SP and Lazarus P (2001) Tobacco carcinogen-detoxifying enzyme UGT1A7 and its association with orolaryngeal cancer risk. Journal of the National Cancer Institute 93:1411-1418.

Zollner G, Marschall HU, Wagner M and Trauner M (2006) Role of nuclear receptors in the adaptive response to bile acids and cholestasis: pathogenetic and therapeutic considerations. Molecular pharmaceutics 3:231-251.

CHAPTER 2

Evaluation of UGT Protein Interactions in Human Hepatocytes:

Effect of siRNA Down Regulation of UGT1A9 and UGT2B7 on

Propofol Glucuronidation in Human Hepatocytes

112 113

Introduction

The UDP-Glucuronosyltransferases (UGTs) are membrane bound proteins localized to the endoplasmic reticulum. This superfamily of enzymes catalyzes the formation of glucuronides by the transfer of glucuronic acid from the co-substrate uridine 5′-diphosphoglucuronic acid (UDPGA) to many endogenous and exogenous substrates, making them more suitable for excretion into the urine or bile (Dutton,

1980; Tukey and Strassburg, 2000). UGTs often work in concert with other key enzymes involved in drug metabolism, such as the cytochrome P450s (CYPs). The

CYPs perform oxidation or reduction reactions to either activate or detoxify the parent compound. These metabolites can then be further detoxified through conjugation reactions, carried out by the UGTs or other Phase II DMEs, including the N- acetyltransferases (NATs), sulfotransferases (SULTs), and glutathione S-transferases

(GSTs). There is accumulating evidence suggesting that protein-protein interactions occur between these enzymes and that these interactions play a significant role in modulating enzyme activity (Fremont et al., 2005; Ikushiro et al., 1997; Ishii et al.,

2010b; Iyanagi, 2007; Kurkela et al., 2003; Lewis et al., 2011; Nakajima et al., 2007;

Operaña and Tukey, 2007; Takeda et al., 2005; Taura et al., 2000). Co-localization and protein-protein interactions between DMEs allows concerted metabolism to occur more efficiently (Ishii et al., 2005). Several studies also suggest that the UGTs themselves dimerize and are functional as dimers in monoglucuronide formation or as tetramers in diglucuronide formation (Gschaidmeier and Bock, 1994). It is the highly variable, substrate binding N-terminus that has generally been implicated in these protein-protein interactions, though evidence exists that the C-terminus may also have

114

a role (Kurkela et al., 2007; Meech and Mackenzie, 1997). Disrupting these interactions has been known to alter Km values and substrate binding specificity (Bock and Kohle, 2009). However, while dimerization has been studied extensively utilizing recombinant systems, it has yet to be confirmed in a physiologically relevant in vitro system. The ability to study UGT protein-protein interactions in human hepatocytes may be valuable in identifying potential disconnects between UGT enzymology in single enzyme versus whole cell systems and in evaluating whether UGT dimerization is a physiologically relevant phenomena or simply an in vitro artifact.

Discovery of RNA interference (RNAi) has allowed for a more complete and systematic analysis of gene expression and function. RNAi is a natural cellular process that controls mRNA levels and serves as a protective mechanism against viral infections. In brief, the process begins with the cleavage of large double-stranded

RNAs (dsRNAs) by Dicer, into small interfering RNAs (siRNAs). Each siRNA is then unwound into two single-stranded RNAs: the passenger strand, which is degraded, and the guide strand, which is incorporated into the RNA-Induced Silencing

Complex (RISC). These siRNAs can then bind to other specific RNAs (mRNA), shutting down mRNA and protein synthesis nonspecifically (McManus and Sharp,

2002). Therefore, introduction of siRNA that targets a gene of interest results in its highly specific and selective down regulation (Rao et al., 2009). In the absence of selective UGT inhibitors, the use of siRNA technology provides a tool to selectively silence individual UGT isoforms, which should allow for the assessment of changes in the enzyme activity both of the targeted UGT as well as other UGTs which may interact on a protein level with the silenced UGT.

115

The objective of this current work was to utilize selective siRNA down regulation to study the effects of UGT1A9-UGT2B7 protein interactions on glucuronidation activity in human hepatocytes. Co-expression of UGT1A9 and

UGT2B7 in HEK cells has previously been shown to enhance the activity of both propofol and morphine glucuronidation when compared to singly expressed systems

(Fujiwara et al., 2010) and as such, UGT1A9 and UGT2B7 represent a rational starting point for the evaluation of UGT protein interactions in human hepatocytes.

Multiple siRNA primers were evaluated and quantitative PCR analysis was used to verify selective down regulation of two UGT isoforms previously shown to be involved in protein-protein interactions. Finally, changes in metabolite formation in hepatocytes treated with siRNA primers were measured by HPLC-MS/MS in order to assess the functional impact of silencing UGT expression on both the UGT isoform of interest as well as on isoforms that may interact with the silenced UGT.

Experimental

Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. Recombinant UGT Supersomes were purchased from BD

Biosciences (San Jose, CA). Cryopreserved plateable human hepatocytes (Donor

4151, Donor 4199, Donor 4237) were purchased from CellzDirect Inc. (Tucson, AZ).

BIOCOAT Cell Environmental Collagen I Cellware 96 well plates were obtained from

BD Biosciences (San Jose, CA). In VitroGRO CP Plating Medium and Torpedo antibiotic mix was purchased from Celsis (Chicago, IL). Silencer Select Predesigned siRNA oligos were obtained from Ambion (Austin, TX). Lipofectamine, Dulbecco’s

116

Modified Eagle Medium (DMEM), maintenance media supplements (100 U/mL penicillin and streptomycin, 6.25 µg/mL insulin, 6.25 µg/mL transferrin, 6.25 ng/mL selenous acid, 1.25 mg/mL bovine serum albumin, 5.35 µg/mL linoleic acid, 2mM

GlutaMAX™, 15mM HEPES, pH 7.4), and RNAiMax Reagent were purchased from

Invitrogen (Carlsbad, CA).

Assessment of Propofol Glucuronidation in Recombinant UGTs. Glucuronidation of propofol was evaluated against recombinantly expressed human hepatic UGT enzymes preparations (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, 2B17). UGT enzymes (0.05 mg) were activated by pre-incubating with alamethicin (25 µg/mg) in 50 mM Tris buffer on ice for 30 min. At the end of the pre-incubation period, incubation mixtures were diluted with purified water and propofol was added to achieve a final concentration of 2.5, 10, or 20 µM. Following a second pre-incubation period (5 min) at 37 ºC, reactions were initiated by addition of UDPGA (1 mM, final concentration) and incubated for 30 min at 37 ºC (100 µL final incubation volume). Control incubations with inactive microsomes (prepared from membranes not expressing UGT enzyme) were treated identically as described above. Reactions were terminated by addition of 200 µL acetonitrile containing formic acid (0.1%, v/v) and 0.1 µM tolbutamide as an internal standard. Following centrifugation (10 min x 1460 g) the resulting supernatants were transferred to 96-well plates and analyzed for the presence of propofol glucuronide by mass spectrometry (LC-MS/MS). Data is plotted as the percent of total glucuronide formation (formation of glucuronide formation by a UGT as a fraction of the total glucuronide formation for all UGTs).

117

Plating and Transfection of Human Hepatocytes. Cryopreserved plateable human hepatocytes from three individual donors (Hu4199, Hu4237, and Hu4151) were placed in a 37°C water bath until thawed and then quickly transferred to fresh, pre-warmed In

Vitro GRO CP media, supplemented with Torpedo antibiotics. Viable cell count was determined using the Trypan Blue exclusion method. Human hepatocytes were then plated on collagen coated, 96-well plates, at a density of 0.7 x 106 viable cells/mL and allowed to attach for 2-4 hours.

For transfection, 25 µM UGT2B7 siRNA (Ambion s14651; 5’ → 3’ sequence,

CATTGAAGAGTAATTAA) and 50 µM UGT1A9 siRNA (Ambion s29248; 5’ → 3’ sequence, CGATCCTTTTGATAACTGT) stock solutions were prepared and each siRNA stock solution was separately added to conical tubes containing OPTIMEM, yielding final siRNA concentrations of 1.0 µM for UGT1A9 oligos and 0.5 µM for

UGT2B7 oligos (siRNA + OPTI). Lipofectamine transfection reagent (TR) (50x) was diluted in a separate conical tube containing OPTIMEM to achieve a 1X solution (TR

+ OPTI). TR + OPTI was added to each siRNA + OPTI mixture (final siRNA concentrations of 0.5 µM for UGT1A9 and 0.25 µM for UGT2B7) and incubated at room temperature for 1 hour with occasional mixing (siRNA + TR + OPTI).

Plating media was removed from hepatocytes and cells were washed with maintenance media (DMEM plus maintenance media supplements), prior to the addition of siRNA + TR + OPTIMEM, giving a final siRNA concentration of 100 nM for UGT1A9 and 50 nM for UGT2B7. Cells were transfected with siRNA for a 72- hour period, which was previously determined to be optimal in regard to transfection

118

efficiency and cellular integrity (data not shown). Cell media was refreshed every 24 hours with pre-warmed maintenance media.

Assessment of siRNA Down Regulation. RNA was isolated using the Ambion Total

RNA Isolation Kit with Applied Biosystems MagMax Express 96 Magnetic Particle

Processor. TaqMan® Probe-Based Gene Expression Analysis was used to quantify siRNA oligo efficiency and specificity. RNA quantity was assessed using the

NanoDrop (Thermo Scientific, Wilmington, DE). All RNA samples were then normalized to 5 ng/µL with nuclease free water. cDNA was synthesized using the

High Capacity cDNA Reverse Transcription kit with a final volume of 20 µL and 66 ng total RNA according to manufacturer’s protocol. After synthesis, the cDNA reactions were diluted to 160 µL total volume with nuclease free water. TaqMan reactions were run on a 7900HT Real-time PCR system in a 384-well optical reaction plate. Each reaction contained 10 µL 2x Gene Expression Master Mix, 5 µL nuclease free water, 1 µL 20x Primer and Probe mix, and 4 µL of cDNA. All reactions were run in duplicate using default cycling parameters. The endogenous control used was

18S1 (Taqman assay ID Hs03928985_g1). Assay ID numbers for the UGT TaqMan assays are as follows: UGT1A1, Hs02511055_s1; UGT1A3, Hs01592480_m1;

UGT1A4, Hs01592480_m1; UGT1A6, Hs01592477_m1; UGT1A9, Hs02516855_sH;

UGT2B4, Hs00607514_mH; UGT2B7, Hs00426592_m1. UGT expression was normalized to expression of the endogenous control 18S1 in each individual well prior to statistical analysis. Control data (100% of control expression) defines the expression of each respective mRNA in the absence of siRNA treatment and after

119

normalization to the 18S1 control. Standard deviations were calculated from the standard deviations of the expression values for both the target UGT of interest and the 18S1 control as noted in equation 1:

(1)

Activity Assays. Following siRNA transfection, hepatocytes were incubated with propofol (0 – 250 µM, final concentration) for 1 hour, conditions which had previously been shown to be linear with respect to incubation time and cell count (data not shown). Propofol was dissolved in DMSO and subsequently diluted into incubation media prior to addition to the hepatocyte cultures in order to maintain a

DMSO concentration of less than 0.1% (v/v). The reaction was then quenched by transferring 100 µL of cell media to a deep well plate containing tolbutamide (0.1 µM) as an internal standard in acetonitrile. Samples were then centrifuged at 3000 rpm for

10 min. and transferred to a second 96-well plate for LC-MS/MS analysis.

Liquid Chromatography/Tandem Mass Spectral Analysis of Glucuronide

Formation. Measurement of propofol glucuronide was performed using LC-MS/MS technology. The LC-MS/MS system consists of an Applied Biosystems 4000 Q-Trap spectrometer (operated in triple quadrupole mode) equipped with an electrospray ionization source (Applied Biosystems, Foster City, CA). The MS/MS system was coupled to two LC-20AD pumps with an in-line CBM-20A controller and DGU-20A5 solvent degasser (Shimadzu, Columbia, MD) and a LEAP CTC HTS PAL autosampler equipped with a dual-solvent self-washing system (CTC Analytics,

120

Carrboro, NC). The injection volume was 20 µl for each analyte. HPLC separation was achieved using a Gemini C18 2.0 × 30 mm 5 µm column (Phenomenex, Torrance,

CA). Gradient elution (flow rate = 500 µl/min) was performed using a mobile phase system consisting of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The gradient conditions were 5% B for 1.0 min, increasing to 100% B from 1.0 to 3.0 min, holding at 100% B from 3.0 to 3.75 min, and returning to 5% B from 3.75 to 5.0 min. HPLC flow was diverted from the MS/MS system for the first

20 s to remove any nonvolatile salts. Generic mass spectrometry parameters included the curtain gas (12 arbitrary units), collision-assisted dissociation gas (medium), ion spray voltage (4500 V), source temperature (500°C), and ion source gas 1 and gas 2

(40 arbitrary units each). Multiple reaction monitoring mass transitions (Q1→Q3) were 352.9→176.9 for propofol glucuronide and 268.9→169.7 for tolbutamide, both utilizing negative ionization. Quantitation of propofol glucuronide was achieved by comparing peak areas in unknown samples to a standard curve of propofol glucuronide from 5 to 2000 ng/mL and weighted using 1/x scaling factor.

Results

Propofol Glucuronidation in Recombinant UGTs

Phenotyping experiments designed to characterize the enzymes responsible for the glucuronidation of propofol at therapeutically relevant concentrations were carried out using UGT Supersomes. At final substrate concentrations of 2.5, 10, or 20 µM, incubations with UGT1A9 accounted for the majority of propofol glucuronide formation in vitro. Incubations with UGT1A6 resulted in a minor amount of propofol

121

glucuronide formation, while those with UGT1A1, UGT1A3, UGT1A4, UGT2B4,

UGT2B7, UGT2B15 or UGT2B17 did not result in the detectable formation of propofol glucuronide (Figure 2-1). Data was normalized to total glucuronide formed for all UGT isoforms.

siRNA Characterization

UGT1A9 and UGT2B7 were chosen for selective siRNA down regulation because these isoforms have been previously implicated in several dimerization studies (Fujiwara et al., 2010). Three oligo sets for each of the two isoforms were tested for down regulation efficiency and selectivity (data not shown). The exon regions and cDNA sequences targeted by the selected oligos are shown in Figure 2-2 and the experimental conditions for the chosen siRNA oligo sets are summarized in

Table 2-1. Quantitative PCR analysis was used to assess relative expression of different UGT isoforms post-transfection and confirmed the successful, selective down regulation of only the desired target gene. In each case, expression of the targeted UGT isoform was reduced to less than 20% of control expression, with minimal changes to the expression of other UGT isoforms (Figure 2-3A, 2-3B).

Individual donor variation in siRNA down regulation was minimal for the three individual donors examined (Figure 2-4A, 2-4B). Though increased variability in the data was observed due to incorporating the standard deviations of both the UGT of interest as well as the 18S1 housekeeping control to calculate the final standard deviation (equation 1), changes in UGT mRNA expression did not reach statistical significance except for the expected isoforms (p < 0.0001).

122

Table 2-1. Summary of the experimental conditions used for siRNA down regulation of UGT1A9 and UGT2B7 in human hepatocytes.

Cell Well Exon siRNA Density Volume Incubation Target Gene RefSeq Targeted (µM) (cells/mL) (µL) Time (hr) UGT1A9 NM_021027.2 1 50 0.7 x 106 100 72 UGT2B7 NM_001074.2 5 25 0.7 x 106 100 72

Table 2-2. Summary of kinetic parameters of propofol glucuronidation in human hepatocytes with and without siRNA treatment.

Vmax (µmol/min) Km (µM) Ki (µM) OPTI Only 0.393 ± 0.009 7.99 ± 0.45 361.7 ± 31.3 TR Only 0.277 ± 0.005 7.62 ± 0.37 521.2 ± 46.3 UGT1A9 siRNA 0.073 ± 0.007 6.02 ± 1.6 448.4 ± 203.5 UGT2B7 siRNA 0.161 ± 0.027 10.5 ± 3.7 198.5 ± 88.9

123

Figure 2-1. Glucuronidation of propofol in recombinant UGTs at therapeutically relevant concentrations. Propofol was primarily glucuronidated by UGT1A9 with a minor contribution from UGT1A6.

124

Figure 2-2. siRNA maps for the selected UGT1A9 and UGT2B7 oligos illustrate the specific regions and consensus sequences (5’ → 3’) targeted for down regulation.

125

Figure 2-3. siRNA down regulation of UGT1A9 and UGT2B7 in human hepatocytes. Quantitative PCR analysis data for transfection with (A) UGT1A9 or (B) UGT2B7 siRNA indicates selective down regulation of the intended target gene. Control data (100% of control expression) defines the expression of each respective mRNA in the absence of siRNA treatment and after normalization to the 18S1 control. An asterisk (*) indicates a statistically significant difference, where p < 0.0001. Error bars indicate the standard deviation for replicate incubations (n=3).

126

Figure 2-4. Individual donor variation in siRNA down regulation was minimal for the three individual donors examined. Quantitative PCR analysis data for transfection with (A) UGT1A9 or (B) UGT2B7 siRNA, indicates selective down regulation of the intended target gene, with minimal variation among hepatocyte donors (Hu4199, Hu4237, and Hu4151). An asterisk (*) indicates a statistically significant difference, where p < 0.001. Error bars indicate the standard deviation for replicate incubations (n=3).

127

Inhibition of Propofol Glucuronidation by siRNA Down Regulation in Human Hepatocytes

To examine the effect of protein-protein interactions on enzymatic activity, enzyme kinetic assays were performed and changes in metabolite formation were measured by LC-MS/MS. siRNA transfected hepatocytes were incubated with increasing concentrations of the UGT1A9 probe substrate propofol (0 – 250 µM, final concentration). Enzyme kinetic parameters are summarized in Table 2-2. Propofol glucuronidation in human hepatocytes was fit to a substrate inhibition model. As expected, propofol glucuronidation was significantly reduced relative to control in hepatocytes treated with UGT1A9 siRNA (Figure 2-5). The effects of silencing

UGT1A9 expression is reflected by a 73.6% reduction in Vmax values for cells transfected with UGT1A9 siRNA (Vmax = 0.073 ± 0.007 µmol/min) compared to controls (OPTI Only Vmax = 0.393 ± 0.009 µmol/min; TR Only Vmax = 0.277 ± 0.005

µmol/min). UGT2B7 down regulation also resulted in a 42.2% reduction in Vmax values for propofol glucuronidation (Figure 2-5; Vmax = 0.160 ± 0.026 µmol/min).

While the Ki values were generally similar for propofol glucuronidation in control hepatocytes and hepatocytes treated with UGT1A9 siRNA, a larger change

(approximately 2.6-fold decrease) was observed in the propofol glucuronidation Ki when the hepatocytes were treated with UGT2B7 siRNA as compared to the transfection reagent control. Km values for propofol glucuronidation were generally unchanged in the presence of either UGT1A9 or UGT2B7 siRNA.

128

Figure 2-5. Inhibition of propofol glucuronidation by siRNA in human hepatocytes. Glucuronidation of the UGT1A9 probe propofol was significantly decreased with down regulation of UGT1A9 expression and to a lesser extent, with down regulation of UGT2B7 expression, compared to control incubations (OPTI Only, TR Only).

Discussion

Glucuronidation is one of the major pathways of metabolism for both endogenous compounds and xenobiotics, accounting for up to 35% of Phase II reactions (Ishii et al., 2010a). With numerous pharmaceuticals such as propofol, irinotecan/SN-38 and opioids as well as many important endogenous compounds such

129

as bilirubin, hormones, and bile acids known to undergo glucuronidation, in vitro systems capable of carrying out glucuronidation reactions have received a significant amount of attention in recent years (Foti and Fisher, 2012; Miners et al., 2004).

The systems currently used for studying glucuronidation in vitro include tissue fractions such as human liver microsomes (HLMs) or S9 fractions, fresh or cryopreserved hepatocytes, and recombinant UGT enzymes. Human liver microsomes are generally considered the easiest to utilize, and contributions from Phase I and

Phase II metabolic enzymes can easily be determined by the selective addition of the necessary cofactors for each pathway (Fisher et al., 2002). Human hepatocytes are the most physiologically relevant in vitro system in which to study glucuronidation activity and generally result in the most accurate prediction of in vivo glucuronidation parameters from in vitro data (Engtrakul et al., 2005; Soars et al., 2002). Cellular systems that over-express one or multiple UGTs of interest have also been used to study glucuronidation phenomena (Coffman et al., 1995; Fujiwara et al., 2007;

Nakajima et al., 2007). Not only can the artificial environments of recombinant systems result in expression levels of the UGTs that may differ from native cells, but also the enzymatic contribution of each UGT isoform is very difficult to isolate, since the interactions vary depending on UGT isoform, substrate, and expression ratio.

Additionally, post-translational modifications to the UGTs, such as phosphorylation and N-glycosylation, that have been shown to impact activity, may not occur in cell expression systems (Ishii et al., 2010a; Miners et al., 2006). Several studies have also demonstrated that variation in lipid composition between preparation and source of synthesis as well as general membrane circumstances contribute to in vitro intrinsic

130

clearances that severely under-predict in vivo hepatic clearance (Fujiwara et al., 2010;

Miners et al., 2004; Soars et al., 2002). Finally, insect-expressed systems, such as

Supersomes, while being a very commonly used phenotyping tool and perhaps the simplest system in which to study a single UGT isoform, lack the potential to exhibit the heterodimeric protein interactions that the more complex systems are capable of exhibiting.

With increasing evidence confirming discrepancies between recombinant systems and whole cell systems, propofol was chosen as a model compound with which to examine potential changes in glucuronidation activity due to protein interactions. While propofol has previously been shown to be a selective substrate for

UGT1A9 (Court, 2005), recent data has indicated the importance of phenotyping compounds at therapeutically relevant concentrations (VandenBrink et al., 2012). As such, a phenotyping assessment of propofol glucuronidation was carried out using concentrations that encompassed the peak plasma concentrations of propofol observed in vivo (Brunton et al., 2006). At the concentrations tested, propofol was selectively glucuronidated by UGT1A9 with only a minor contribution from UGT1A6, indicating that it was an appropriate choice of model substrates with which to examine UGT activity in more complex in vitro systems.

To date, the use of siRNA to examine drug metabolizing enzyme activity is fairly limited. The technology is more commonly used to target the expression of over expressed molecular targets in cancer therapy or to discern the importance of an enzymatic pathway in an in vitro pharmacology assay (Rao et al., 2009). The use of siRNA to study glucuronidation has been previously reported in both HeLa cells as

131

well as in a Caco-2 cell system (Jiang et al., 2012; Liu et al., 2007). Upon down regulation of UGT1A6 expression in Caco-2 cells, a significant decrease in the glucuronidation of apigenin was observed, resulting in UGT1A6 being implicated as the primary UGT isoform involved in the glucuronidation of flavanoids in Caco-2 cells.

Previous investigations into the interactions between human UGT2B7 and

UGT1A enzymes demonstrated that co-expression of UGT2B7 with UGT1A9 in HEK cells resulted in enhancement of propofol glucuronidation, in comparison with the

UGT1A9 single expression system (Fujiwara et al., 2010). Using siRNA inhibition in human hepatocytes, we have shown that down regulation of UGT2B7 expression also results in a decrease in the glucuronidation rate of propofol, presumably due to disruption of protein interactions between UGT1A9 and UGT2B7. While Km values remained relatively unchanged when UGT2B7 was targeted, decreases in both the observed Ki and Vmax values for propofol glucuronidation (fit to a substrate inhibition kinetic model) were observed as compared to the transfection reagent control. As Ki is the dissociation constant for an inhibitory enzyme-substrate complex, any modifications to protein structure or conformation could be expected to result in changes to the binding affinity for the inhibitory ligand in the UGT1A9 active site

(Segel, 1975). As such, the more tightly bound inhibitor (as defined by the lower Ki value) may account for some of the observed decrease in UGT1A9 activity when

UGT2B7 is silenced in the hepatocyte incubations. In addition, it has previously been shown that UGTs form catalytically active dimers through interactions of their amino- terminal domains, an interaction which serves to stabilize the resulting protein

132

complex (Meech and Mackenzie, 1997). Conversely, the lack of such an interaction could conceivably serve to destabilize the UGT1A9 protein in hepatocytes pre-treated with UGT2B7 siRNA, resulting in the observed decrease in Vmax values for propofol glucuronidation when UGT2B7 is down regulated. One final scenario that must be considered is that the incorporation of UGT2B7 siRNA may affect the translation or protein folding properties of UGT1A9, a phenomenon that we are currently investigating.

In summary, the data presented in this manuscript supports the utility of siRNA down regulation as an important process for evaluating UGT enzymology and suggests that UGT protein interactions are a physiologically relevant phenomena whose effects can be observed in human hepatocytes. The data also confirm previous interactions noted for UGT1A9 and UGT2B7 in over-expressed cellular systems.

While single UGT expression systems will continue to be a useful tool both in characterizing UGTs and phenotyping drugs that undergo glucuronidation, the current data support the caution that should be taken in utilizing in vitro UGT systems in which heterodimeric protein interactions are unable to occur.

Chapter 2, in full, is a currently being prepared for submission in Archives of

Biochemistry and Biophysics, 2012, Dickmann L., Tracy J., Tukey R.H., Wienkers

L.C., and Foti R.S. I was the primary investigator and author of this paper.

References

Bock KW and Kohle C (2009) Topological aspects of oligomeric UDP- glucuronosyltransferases in endoplasmic reticulum membranes: advances and open questions. Biochemical pharmacology 77:1458-1465.

133

Brunton L, Lazo J and Parker K (2006) Goodman & Gilman's The Pharmacological Basis of Therapeutics, Eleventh Edition, McGraw-Hill Professional, New York.

Coffman BL, Green MD, King CD and Tephly TR (1995) Cloning and stable expression of a cDNA encoding a rat liver UDP-glucuronosyltransferase (UDP- glucuronosyltransferase 1.1) that catalyzes the glucuronidation of opioids and bilirubin. Mol Pharmacol 47:1101-1105.

Court MH (2005) Isoform-selective probe substrates for in vitro studies of human UDP-glucuronosyltransferases. Methods Enzymol 400:104-116.

Dutton GJ (1980) Acceptor substrates of UDP-glucuronosyltransferase and their assay, in Glucuronidation of Drugs and Other Compounds (Dutton GJ ed) pp 69-78, CRC Press, Boca Raton.

Engtrakul JJ, Foti RS, Strelevitz TJ and Fisher MB (2005) Altered AZT (3'-azido-3'- deoxythymidine) glucuronidation kinetics in liver microsomes as an explanation for underprediction of in vivo clearance: comparison to hepatocytes and effect of incubation environment. Drug Metab Dispos 33:1621-1627.

Fisher MB, Jackson D, Kaerner A, Wrighton SA and Borel AG (2002) Characterization by liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry of two coupled oxidative-conjugative metabolic pathways for 7-ethoxycoumarin in human liver microsomes treated with alamethicin. Drug Metab Dispos 30:270-275.

Foti RS and Fisher MB (2012) UDP-Glucuronosyltransferases: Pharmacogenetics, Functional Characterization, and Clinical Relevance, in Encyclopedia of Drug Metabolism and Interactions (Lyubimov AV ed), John Wiley & Sons, Inc.

Fremont JJ, Wang RW and King CD (2005) Coimmunoprecipitation of UDP- glucuronosyltransferase isoforms and cytochrome P450 3A4. Mol Pharmacol 67:260- 262.

Fujiwara R, Nakajima M, Oda S, Yamanaka H, Ikushiro S, Sakaki T and Yokoi T (2010) Interactions between human UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A enzymes. J Pharm Sci 99:442-454.

Fujiwara R, Nakajima M, Yamanaka H, Katoh M and Yokoi T (2007) Interactions between Human UGT1A1, UGT1A4, and UGT1A6 Affect Their Enzymatic Activities. Drug Metabolism and Disposition 35:1781-1787.

Gschaidmeier H and Bock KW (1994) Radiation inactivation analysis of microsomal UDP-glucuronosyltransferases catalysing mono- and diglucuronide formation of 3,6-

134

dihydroxybenzo(a)pyrene and 3,6-dihydroxychrysene. Biochemical pharmacology 48:1545-1549.

Ikushiro S, Emi Y and Iyanagi T (1997) Protein-protein interactions between UDP- glucuronosyltransferase isozymes in rat hepatic microsomes. Biochemistry 36:7154- 7161.

Ishii Y, Nurrochmad A and Yamada H (2010a) Modulation of UDP- glucuronosyltransferase activity by endogenous compounds. Drug Metab Pharmacokinet 25:134-148.

Ishii Y, Takeda S and Yamada H (2010b) Modulation of UDP-glucuronosyltransferase activity by protein-protein association. Drug Metab Rev 42:145-158.

Ishii Y, Takeda S, Yamada H and Oguri K (2005) Functional protein-protein interaction of drug metabolizing enzymes. Front Biosci 10:887-895.

Iyanagi T (2007) Molecular mechanism of phase I and phase II drug-metabolizing enzymes: implications for detoxification. Int Rev Cytol 260:35-112.

Jiang W, Xu B, Wu B, Yu R and Hu M (2012) UDP-Glucuronosyltransferase (UGT) 1A9-Overexpressing HeLa Cells Is an Appropriate Tool to Delineate the Kinetic Interplay between Breast Cancer Resistance Protein (BRCP) and UGT and to Rapidly Identify the Glucuronide Substrates of BCRP. Drug Metabolism and Disposition 40:336-345.

Kurkela M, Garcia-Horsman JA, Luukkanen L, Morsky S, Taskinen J, Baumann M, Kostiainen R, Hirvonen J and Finel M (2003) Expression and characterization of recombinant human UDP-glucuronosyltransferases (UGTs). UGT1A9 is more resistant to detergent inhibition than other UGTs and was purified as an active dimeric enzyme. J Biol Chem 278:3536-3544.

Kurkela M, Patana AS, Mackenzie PI, Court MH, Tate CG, Hirvonen J, Goldman A and Finel M (2007) Interactions with other human UDP-glucuronosyltransferases attenuate the consequences of the Y485D mutation on the activity and substrate affinity of UGT1A6. Pharmacogenetics and genomics 17:115-126.

Lewis BC, Mackenzie PI and Miners JO (2011) Homodimerization of UDP- glucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling. Biochemical pharmacology 82:2016-2023.

Liu X, Tam VH and Hu M (2007) Disposition of Flavonoids via Enteric Recycling: Determination of the UDP-Glucuronosyltransferase Isoforms Responsible for the Metabolism of Flavonoids in Intact Caco-2 TC7 Cells Using siRNA. Molecular Pharmaceutics 4:873-882.

135

McManus MT and Sharp PA (2002) Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 3:737-747.

Meech R and Mackenzie PI (1997) UDP-glucuronosyltransferase, the role of the amino terminus in dimerization. J Biol Chem 272:26913-26917.

Miners JO, Knights KM, Houston JB and Mackenzie PI (2006) In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises. Biochemical pharmacology 71:1531-1539.

Miners JO, Smith PA, Sorich MJ, McKinnon RA and Mackenzie PI (2004) Predicting human drug glucuronidation parameters: application of in vitro and in silico modeling approaches. Annu Rev Pharmacol Toxicol 44:1-25.

Nakajima M, Yamanaka H, Fujiwara R, Katoh M and Yokoi T (2007) Stereoselective glucuronidation of 5-(4'-hydroxyphenyl)-5-phenylhydantoin by human UDP- glucuronosyltransferase (UGT) 1A1, UGT1A9, and UGT2B15: effects of UGT-UGT interactions. Drug Metab Dispos 35:1679-1686.

Operana TN and Tukey RH (2007) Oligomerization of the UDP- glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation. J Biol Chem 282:4821-4829.

Rao DD, Vorhies JS, Senzer N and Nemunaitis J (2009) siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev 61:746-759.

Segel I (1975) Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems, John Wiley & Sons, Inc, New York.

Soars MG, Burchell B and Riley RJ (2002) In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance. J Pharmacol Exp Ther 301:382-390.

136

Tukey RH and Strassburg CP (2000) Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 40:581-616.

VandenBrink BM, Foti RS, Rock DA, Wienkers LC and Wahlstrom JL (2012) Evaluation of CYP2C8 Inhibition In Vitro: Utility of Montelukast as a Selective CYP2C8 Probe Substrate. Drug Metabolism and Disposition 39:1546-1554

CHAPTER 3

The Regulatory Role of Oral Arsenic in hUGT1 Mice

137 138

Introduction

Humanized Mice as a Sensor for Environmental Toxicant Exposure

The humanized mouse model that was utilized in this dissertation study was created by crossing heterozygous TgUGT1 mice with heterozygous Ugt1-null mice.

To generate the TgUGT1 model, a BAC clone carrying the entire hUGT1 locus was microinjected into the pronucleus of a fertilized mouse egg and subsequently transplanted into the oviduct of a pseudo-pregnant female. Breedings were then carried out to establish heterozygous TgUGT1 founders. Characterization of the

TgUGT1 line confirmed that the human UGT1 locus was successfully expressed, with all 9 UGT1A genes being regulated in a pattern concordant with the tissue specific expression profiles previously documented in humans (Chen et al., 2005). The most dramatic expression of the UGT1 locus and the UGT1A1 gene was observed in the GI tract of the TgUGT1 mice (Senekeo-Effenberger et al., 2007; Chen et al., 2005) and occurred in patterns similar to those seen in the human GI tract (Strassburg et al.,

2000; Strassburg et al., 1999).

To be able to compare and contrast the role of human glucuronidation in disease and toxicity, the human UGT1 locus needed to be expressed in an Ugt1-null background. The Ugt1-null model was created by inactivation of the entire murine

Ugt1 locus through targeted interruption of the Ugt1 gene. This was accomplished by introducing a genetic lesion into conserved exon 4 (Nguyen et al., 2008). Interruption of the conserved region of the locus leads to a complete absence of Ugt1a RNA and protein and the inability to metabolize bilirubin. Thus, deletion of the murine Ugt1 gene leads to the accumulation of toxic levels of unconjugated bilirubin in the serum,

139

evident early after birth by an orange tint of the skin, which eventually becomes lethal within 7 days after birth. Crossing the TgUGT1 mice with the Ugt1-null mice results in the generation of the Tg(UGT1)Ugt1-/- mice, which represent the fully humanized

UGT1 (hUGT1) model (Fujiwara et al., 2010b). hUGT1 mice do not exhibit the developmental lethality observed in the Ugt1-null model that occurs in the absence of the Ugt1a1 gene.

The regulatory region of the human UGT1A1 gene is known as the PBREM and is where xenobiotic receptors, such as PXR, CAR, and PPARα, as well as the environmental sensors, such as AhR, and the antioxidant receptor Nrf2, can associate with DNA to induce transcription, thus playing a key role in the in vivo regulation of

UGT1A1. Since numerous environmental toxicants can induce UGT1A1 through association with these xenobiotic receptors and with UGT1A1 being the sole enzyme that can metabolize bilirubin, induction of UGT1A1 directly leads to reduction of serum bilirubin levels. It has been shown that treatments of hUGT1 mice with TCDD and PB, which are AhR and CAR agonists, dramatically induce UGT1A1 expression, thus reducing their serum bilirubin levels (Fujiwara et al., 2010b; Fujiwara et al.,

2012). Treatment of hUGT1 mice with LPS can also induce UGT1A1 expression and reduce serum bilirubin by generating oxidative stress (Fujiwara et al., 2012). These findings indicate that hUGT1 mice are responsive to toxic substances and have potential to serve as an in vivo biosensor for environmental toxicant exposure.

140

Environmental Arsenic Contamination

Environmental arsenic contamination is a significant problem worldwide, with drinking water being the most common source of exposure. This naturally occurring metalloid is ubiquitously distributed throughout Earth's crust, although typically complexed with pyrite. Under certain conditions it can dissociate and enter groundwater. Arsenic contamination of drinking water can also be attributed to certain anthropogenic sources, including agricultural and industrial practices

(Kumagai and Sumi, 2007). Additionally, some crops and organisms tend to bioaccumulate arsenic and therefore regional and individual eating habits can greatly affect dietary arsenic intake (Bernstam and Nriagu, 2000). There is extensive epidemiological evidence linking arsenic exposure to various diseases

(arteriosclerosis, cardiovascular disease, Blackfoot disease) (Navas-Acien et al.,

2005), numerous cancers (skin, bladder, lung, liver, prostate, kidney) (Kligerman and

Tennant, 2007; Chen et al., 1992), diabetes (Diaz-Villasenor et al., 2007), and certain neurological ailments (Alzheimer’s and Parkinson’s) (Schmuck et al., 2005; Vahidnia et al., 2007). Arsenic absorption and toxicity greatly depends on the form in which it is ingested (Bernstam and Nriagu, 2000). Soluble inorganic species (sodium arsenite and sodium arsenate) have been determined as most readily absorbed from the GI tract, with typical absorption rates being 40–100% of the ingested amount (Pontius et al., 1994). The predominant form of arsenic found in drinking water is arsenate

(As5+), but it is readily reduced in vivo to the more toxic species (As3+) by either glutathione or arsenate reductase (Bode and Dong, 2002). Subsequent elimination of the trivalent species from the body occurs through reduction, methylation, and

141

glutathione conjugation, to yield polar metabolites that are substrates for transporters

(Kumagai and Sumi, 2007). However, very little is known about the effects of arsenic exposure on human UGT1A1 and the molecular mechanisms involved in UGT regulation by arsenic. This is of great concern, considering how prominent arsenic exposure has recently become in the news. Scientific articles and influential news websites have been bringing to light the presence of high levels of arsenic in many commonly consumed and everyday items, including apple and grape juice (Cohen,

2011), chicken-feed (Fairbrother, 2012) and many baby formulas, and rice cereals made from contaminated rice flour (Carroll, 2012). As of September 20, 2012, levels of arsenic in rice have skyrocketed, ultimately urging the FDA to define standards to protect consumers (Garber, 2012). Moreover, a FoxNews.com article from June 2012 has discussed the EPA’s decision to decrease the arsenic standard for drinking water from 10 ppb to 2 -3 ppb, as 10 ppb has just been shown to stimulate adverse health effects in pregnant and lactating female mice as well as their offspring (Kozul-Horvath et al., 2012; Grush, 2012). The shocking and on-going prevalence of contamination begs the question: what effect does arsenic have on important biological processes, such as xenobiotic metabolism?

Arsenic is known to alter multiple cellular pathways, including expression of growth factors, suppression of cell cycle checkpoint proteins, promotion of and resistance to apoptosis, inhibition of DNA repair, alterations in DNA methylation, decreased immunosurveillance, and increased oxidative stress, by disturbing the pro/antioxidant balance (Flora, 2011). This extensive list of potential targets explains the wide range of resulting disease manifestations due to exposure, such as

142

carcinogenicity, genotoxicity, diabetes, cardiovascular, and nervous systems disorders.

However, in 1998, Hu et al. analyzed the dose-response for arsenic inhibition of several purified human DNA repair enzymes, including DNA polymerase beta, DNA ligase I, and DNA ligase III. It was observed that most enzymes, even those with critical thiol groups, were surprisingly insensitive to arsenite and that only a few sensitive enzymes were responsible for arsenic-induced cellular toxicity (Hu et al.,

1998). In addition, arsenic concentrations required to deactivate certain enzymes have been determined to be much lower than what is required for direct binding to thiol groups. This was documented by Samikkannu et al. in 2003, who observed inactivation of pyruvate dehydrogenase activity by As2O3 to be about 38 times more potent in a human leukemia cell line (HL60) than in pure enzyme preparation, suggesting that while As2O3 inactivates activity in pure enzyme preparation by binding to the dithiols, this same mechanism may be different in HL60 cells. The IC50 values for As2O3 and phenylarsine oxide (PAO) to decrease the cellular vicinal thiol content of HL60 cells were determined to be 80.0 µM and 1.9 µM, respectively, confirming

As2O3 as a weak thiol reacting agent in comparison to PAO. Dithiol compounds were also capable of suppressing PAO inhibition of pyruvate dehydrogenase activity but not for As2O3, and antioxidants suppressed As2O3 inhibition of pyruvate dehydrogenase activity but not for PAO. Lastly, As2O3 increased cellular H2O2 production in HL60 cells, while PAO did not, Fenton metal chelators decreased and Fenton metals increased As2O3 inhibition of pyruvate dehydrogenase activity, and HL60 treatment with H2O2 and Fenton metals decreased the pyruvate dehydrogenase activity, ultimately implicating inactivation of pyruvate dehydrogenase via ROS-mediated

143

mechanisms (Samikkannu et al., 2003). These findings suggest that arsenic-induced chromosomal damage and inhibition of DNA repair are not the result of direct enzyme inhibition, but instead an indirect effect caused by exposure-induced changes in cellular redox levels or alterations in signal transduction pathways with subsequent changes in gene expression (Hu et al., 1998; Bernstam and Nriagu, 2000).

Arsenic-Induced Generation of Reactive Oxygen Species

Certain metals, including arsenic, have been reported to be potent carcinogenic or toxic agents in both humans and animals. While it is known that arsenic causes

DNA damage and lipid peroxide formation in vitro and in vivo, the underlying molecular mechanisms of its toxicity have eluded scientists. However, what is well established is that reactive oxygen species (ROS), such as superoxide anion (O2-),

. 1 hydroxyl radical ( OH), singlet oxygen (O2), and hydrogen peroxide (H2O2), generated by arsenic exposure have a wide potential for causing cellular injury and numerous toxic effects (Menghini, 1988; Ercal et al., 2001), and thus may at least partly be contributing to the process of arsenic-induced toxicity (Sugiyama, 1994).

Aerobic organisms have evolved a sophisticated regulatory system by which to defend against oxidative damage. Overproduction or accumulation of oxygen free radicals in cells, termed oxidative stress, can damage DNA, proteins, lipids and other molecules (Cerutti, 1985; Lee and Ho, 1995). It therefore follows that these oxidative processes could potentially play important roles in arsenic-induced damage (Klein et al., 1991; Sugiyama, 1994). The first oxidative stress theory, fueled by the documentation of arsenic-induced free radical formation, was presented in 1989 by

144

Yamanaka et al. In vitro experiments revealed diminished trivalent dimethylarsine induced DNA strand breaks upon addition of superoxide dismutase (SOD) and catalase (CAT), suggesting that reactive oxygen produced by exposure was involved in the induction of DNA damage (Yamanaka et al., 1990; Yamanaka et al., 1989;

Kitchin, 2001). It was later confirmed that toxicity resulted from molecular oxygen reacting with dimethylarsine to form the dimethylarsinic radical and superoxide anion, and subsequent addition of another molecule of molecular oxygen formed the dimethylarsinic peroxyl radical that is detrimental to cells. Generation of O2- and

H2O2 post arsenic exposure has been demonstrated in various human cell lines, including human vascular smooth muscle cells (Lynn et al., 2000), human–hamster hybrid cells (Liu et al., 2001), and vascular endothelial cells (Barchowsky et al.,

1999), although HEL30 (Corsini et al., 1999), NB4 (Jing et al., 1999), and CHOK1

(Wang et al., 1996) have only shown induction of H2O2. Arsenic-induced hydroxyl radical generation has also been reported in the rat brain (Garcia-Chavez et al., 2003).

While there is a plethora of direct evidence of arsenic induced ROS, reported indirect evidence also demonstrates arsenic’s effect on the cellular antioxidant defense system.

Low molecular weight donors of SH groups and enzymes, which can catalyze the reduction of SH groups in proteins and detoxify pro-oxidants by conjugation with glutathione, are part of the system that helps regulate the redox status of cellular thiols and protect SH-containing proteins from excessive oxidation (Samikkannu et al.,

2003). Arsenic affects many oxygen-radical-scavenging enzymes called antioxidant enzymes, such as SOD, CAT, glutathione peroxidase (GPx), GST, and glutathione reductase (Flora, 2011). Increases in protective enzymes, CAT and SOD, have been

145

shown to suppress arsenic-induced sister chromatid exchanges in human lymphocytes

(Nordenson and Beckman, 1991). Arsenic treatment of an X-ray sensitive Chinese hamster ovary cell line (XRS-5), which is sensitive to several free-radical generating agents, including H2O2, exhibited arsenic hypersensitivity, thus implicating that the genotoxicity of arsenite was mediated by ROS (Wang and Huang, 1994). In 1995,

Lee et al. studied sodium arsenite induced oxidative stress in human fibroblasts. They observed increased formation of fluorescent dichlorofluorescein (DCF) by oxidation of the nonfluorescent form, which was also inhibited by a radical scavenger, confirming arsenic’s ability to alter cellular redox levels. Treatment of human fibroblasts also revealed significantly increased heme oxygenase (HO-1) activity, ferritin level, glutathione levels and SOD activity, slightly decreased GPx activity, significantly decreased CAT activity, and no effect on glucose-6-phosphate dehydrogenase (G6PD) activity (Lee and Ho, 1995). These results demonstrate the effect of sodium arsenite on cellular antioxidant activities that can lead to enhanced oxidative stress in HFW. In 2000, Maiti and Chattetjee also reported tissue-specific protective mechanisms against arsenic exposure in male Wistar rats. The kidneys were observed to be more vulnerable to arsenic in rats exposed to 3.33mg/kg sodium arsenite per day for 14 days and displayed significant increases in lipid peroxidation and decreased SOD and CAT activities. Lipid peroxidation and SOD activity in liver remained unchanged post treatment due to significantly increased glutathione levels and protection of activities of glutathione reductase and GST from arsenite-induced oxidative damage by some antioxidant components, such as glutathione, GST, and

G6PD (Maiti and Chatterjee, 2000). These findings clearly support that arsenic

146

exposure results in ROS generation in various cellular systems. Since 1989, there has been increasing evidence supporting oxidative stress theory and greater scientific acceptance of this as a significant mode of action. As such, oxidative stress is currently the most widely accepted and studied mechanism of arsenic toxicity (Ercal et al., 2001).

Arsenic Can Modulate Xenobiotic Transcriptional Activation

The UGT1A1 gene contains a series of xenobiotic receptor enhancer sequences that recognize LXRα (unpublished observations), AhR (Yueh et al., 2003; Bonzo et al., 2007), CAR (Sugatani et al., 2001; Sugatani et al., 2005b), PXR (Xie et al., 2003),

GR (Sugatani et al., 2005a), PPARα (Senekeo-Effenberger et al., 2007), and Nrf2

(Yueh and Tukey, 2007) and is one of the few genes that can be independently regulated by activation of any one of these transcriptional factors. Therefore, there are numerous xenobiotics and environmental toxicants that can induce UGT1A1 gene expression. However, very little is known with regards to how arsenic modulates

UGT gene expression. Evidence of transcriptional regulation of other DMEs by arsenic can help provide guidance for elucidating mechanisms involved in arsenic- induced UGT expression.

There is emerging evidence that heavy metals regulate CYP1A1 activity by enhancing AhR in a metal and species-dependent manner (Wu et al., 2009). Previous reports have confirmed arsenic’s ability to alter CYP activities. Increases in B[a]P metabolites and BPDE adducts were observed in animals co-exposed to B[a]P and arsenic (Evans et al., 2004). Since CYP1 enzymes, specifically CYP1A1, are known

147

to be key in B[a]P metabolism (Shimada et al., 2002), Wu et al. postulated in 2009 that these observed increases in B[a]P metabolism with arsenic exposure could be due to enhanced CYP1A1 expression and activity (Wu et al., 2009). They were able to successfully demonstrate increased CYP1A1 mRNA expression in a human lung adenocarcinoma cell line treated with arsenic, as well as increased CYP1A1 expression and activity in lung tissues of arsenic-exposed mice. Elevated CYP1A1 expression was also effectively blocked with an AhR antagonist, indicating that arsenic-induced CYP1A1 expression and activity occurred via AhR activation. In addition, arsenic-induced AhR activation and CYP1A1 expression were both increased with pro-oxidant treatment and suppressed by antioxidants, such as N-acetylcysteine

(NAC) and catalase. These findings illustrate that arsenic exposure enhances

CYP1A1 expression and activity through AhR activation (Wu et al., 2009). With

UGT1A1 being similarly regulated by AhR, it can therefore follow that arsenic may be capable of regulating UGT1A1 gene expression.

The xenobiotic nuclear receptors PXR and CAR have been well established as xenobiotic sensors (Xie et al., 2003; Blumberg et al., 1998; Xie et al., 2000). Arsenic- treated Tg-CYP3A4 mice were shown to exhibit elevated hepatic CYP expression levels as well as increased PXR and RXR mRNA, which heterodimerize to regulate gene expression of several Phase I and II DMEs (Falkner et al., 2001). However, when Noreault et al. investigated whether arsenite decreased CYP3A4 induction by

PB or rifampicin, which is capable of inducing CYP3A4 either through CAR or PXR

(Goodwin et al., 2002a; Goodwin et al., 2002b), the authors observed that treatment of human hepatocytes with arsenite in the presence of CYP3A4 inducers, PB or

148

rifampicin caused major decreases in CYP3A4 mRNA, protein, and activity.

CYP3A4 in untreated cells was also decreased following arsenite treatment. Since transcription of CYP3A4 is primarily regulated by heterodimers of RXR and PXR, the authors investigated the affect of arsenite on PXR and RXR expression. Arsenite failed to affect expression of PXR, yet caused marked decreases in PXR responsiveness to rifampicin. In addition, arsenite caused a large decrease in nuclear

RXR protein and to a lesser extent in RXR mRNA. These findings suggest that arsenite inhibits both untreated and induced CYP3A4 transcription in primary human hepatocytes by decreasing the activity of PXR, as well as RXR expression (Noreault et al., 2005). Since CAR and PXR are similar in that they form heterodimers with RXR, arsenic exposure that alters RXR expression can subsequently affect transcriptional activation of target genes. It can therefore be hypothesized that since the UGT1A1 gene contains CAR and PXR enhancer sequences, UGT1A1 gene expression can also be impacted by arsenic exposure.

Nrf2 is a redox-sensitive transcription factor that regulates the expression of genes encoding antioxidants and xenobiotic detoxification enzymes for cytoprotection against oxidative stress and xenobiotics (Singh et al., 2006; Itoh et al., 1995; Ishii et al., 2000). In response to oxidative stress or direct xenobiotic stimulation, Nrf2 dissociates from Keap1 and enters the nucleus where it functions as a strong transcriptional activator. Arsenic has been shown to activate Nrf2, which in turn regulates the expression of genes encoding for many antioxidative response enzymes

(Pi et al., 2003). Upregulation of Nrf2 has been detected in an immortalized, non- tumorigenic human keratinocyte cell line (HaCaT) that was continuously exposed to

149

environmentally relevant levels of inorganic arsenite for 28 weeks (Pi et al., 2008). It has also been demonstrated that As3+ can activate Nrf2 by directly binding Keap1 (He et al., 2006; He and Ma, 2010). Most importantly, our lab has previously documented that UGT1A1 in hUGT1 mice is regulated by the cellular antioxidant sensor Nrf2 following activation in response to ROS, and Nrf2 subsequently targets the antioxidant response element (ARE) in the promoter region of the UGT1A1 gene (Yueh and

Tukey, 2007). Since regulation of the UGT1A1 gene by Nrf2 is sensitive to changes in

ROS, Nrf2 may control those events that regulate expression of intestinal UGT1A1 in response to arsenic exposure.

Arsenic Impacts the NF-κB/IKK Signaling Pathway

Arsenic causes oxidative stress, which in turn, activates signaling pathways involved in the regulation of early response genes, such as NF-κB, to respond to alterations in the intracellular redox status (Felix et al., 2005; Kapahi et al., 2000).

Stress response transcription factors are particularly important in these early responses, as they regulate the expression of a variety of downstream target genes involved in cellular antioxidant defense mechanisms (Kapahi et al., 2000). However, the molecular mechanisms by which arsenic affects the NF-κB pathway have yet to be identified, since controversial studies have shown that arsenic-induced changes in gene expression can occur by either suppression (Jeong et al., 2004) or activation

(Barchowsky et al., 1996; Kitamura and Hiramatsu, 2010) of NF-κB, leading to the downregulation or upregulation of downstream genes, respectively. Arsenic-induced generation of ROS has been shown to activate the NF-κB (Baldwin, 2001) and NF-κB

150

dependent gene transcription pathways, including NO, AP-1, p53, and p21 (Buzard and Kasprzak, 2000). Population studies of newborns whose mothers were exposed to varying levels of arsenic revealed differential expression of stress response and cell cycle regulatory genes (Fry et al., 2007). In addition, it is widely known that NF-κB regulates genes encoding for cytokines, cytokine receptors, cell adhesion molecules, and growth regulators (Baldwin, 2001). As such, these findings implicate several avenues by which arsenic can modify cell cycle control, proliferation, and cellular morphology.

IκB phosphorylation and degradation has been identified as the most likely signaling step affected by oxidative stress (Li and Karin, 1999). Arsenic has been shown to exert its biological effects by reacting with IKK’s free thiol to inhibit NF-κB signaling (Roussel and Barchowsky, 2000). Blocked IKK results in limited degradation of IκB and decreased NF-κB activation (Kapahi et al., 2000). In contrast, very early research initially proposed ROS-induced oxidative stress as a universal mechanism for NF-κB activation by diverse agents, including arsenic (Schreck et al.,

1992b). Simply exposing various cell lines to H2O2 has provided direct evidence that

ROS are capable of regulating NF-κB (Sen and Packer, 1996; Schreck et al., 1991;

Manna et al., 1998; Li and Karin, 1999). However, other researchers were unable to detect NF-κB activation by H2O2 in Hela, HEK293, fibroblast, or Jurkat T cells, implicating H2O2-induced NF-κB activation as highly cell specific and that H2O2 is not a likely mediator of activation (Anderson et al., 1994). Nonetheless, there still is extensive evidence that implicates reactive oxidative intermediates as NF-κB- activating signals, including the inhibition of NF-κB activation by various antioxidants

151

and by overexpression of antioxidant enzymes (Schreck et al., 1992a). Additionally, activation of NF-κB by arsenic trioxide (As2O3) at non-cytotoxic levels has been demonstrated in studies from several groups via gel shift assays by which to monitor activation and nuclear translocation of NF-κB and NF-κB-dependent reporter gene assays to indicate NF-κB activity (Kaltreider et al., 1999; Chen and Shi, 2002a). It has also been recently demonstrated through Chen and Shi’s research that arsenic is capable of activating NF-κB through the MAPKs. Their studies using wild-type and stress-activated protein kinase (SAPK)/ERK kinase (sek1) gene knockout mouse embryo stem cells suggested that As3+-induced NF-κB occurred through a signaling pathway that involved SEK1 (MKK4)-JNK (Chen and Shi, 2002b) and that neither

ERK nor p38 was required for As3+-induced NF-κB activation. In contrast, blocked

NF-κB activation was observed due to inhibition of ERK with either the specific inhibitor, PD98059, or in cells deficient of Erk, showing that ERK is required for NF-

κB activation in mouse skin epidermal JB6 cells (Huang et al., 2001). These findings implicate the potential for cross talk between the MAPKs and NF-κB/IKK signaling pathways in response to arsenic-induced oxidative stress, which adds to the complication of elucidating the mechanisms involved in arsenic exposure.

Previous work in our lab has confirmed activation of NF-κB/IKK signaling in

UGT1A1 induction with stress-inducing agents, LPS and cadmium (Fujiwara et al.,

2012). Since humanized neonatal UGT1 mice treated with these NF-κB activators exhibited marked intestinal UGT1A1 induction that resulted in significant decreases in bilirubin levels, it is reasonable to consider that other metal contaminants, such as arsenic, can regulate UGT1A1 in a similar fashion (Fujiwara et al., 2012). The role of

152

arsenic in regulating UGT1A1 expression, specifically in small intestine, via the NF-

κB/IKK pathway has yet to be explained.

Arsenic Influences the MAPK Signaling Pathway

The MAPKs transmit extracellular signals to induce expression of various genes that mediate cell apoptosis, differentiation, proliferation, and transformation. Of the three major classes of MAPKs, ERKs are mainly involved in growth factor- induced cell differentiation, proliferation, and transformation signaling, while JNK and p38 mediate cytokine and numerous stress-induced cell response, cell growth arrest, and apoptosis (Qian et al., 2003). Several environmental contaminants have been shown to induce apoptosis, or programmed cell death, in Hepa1c1c7 cells (Lei et al., 1998), Daudi human B cells (Salas and Burchiel, 1998), human ectocervical cells

(Rorke et al., 1998), and A20.1 murine B-cells (Burchiel et al., 1993). In a study assessing particulate matter (PM)-induced apoptosis in RAW 264.7 macrophage cells, carbon black particles containing B[a]P were shown to stimulate the release of TNF-α, a known MAPK initiator. Furthermore, cells treated with a MAPK kinase inhibitor did not undergo apoptosis, indicating that the MAPK pathway plays an important role in regulating PM- and TNF-α-induced apoptosis in this cell culture model (Chin et al.,

1998). In addition, B[a]P has been shown to activate JNK1 and induce caspase-3- mediated apoptosis in Hepa1c1c7 cells as well as upregulate α-PAK-exchange factor, which is upstream of JNK, in a manner concordant with activation of JNK1 in both

HEK293 and HeLa cells (Yoshii et al., 2001).

The cell deals with the onset of oxidative stress by programming the regulation

153

of genes to protect against the potential hazards of ROS. As such, the JNK MAPKs have been identified as an important family of stress activated protein kinases that are regulated through complex signal transduction cascades and in turn phosphorylate and regulate the activity of various transcriptional factors. The JNK subfamily of MAPKs is activated in response to a variety of extracellular stimuli, including mitogens, pro- inflammatory stimuli, nutrients, and environmental stimuli or changes that induce oxidative stress (Davis, 2000; Ip and Davis, 1998). JNK was originally identified by its ability to bind (Kyriakis and Avruch, 1990; Adler et al., 1992; Hibi et al., 1993) and phosphorylate c-Jun (Pulverer et al., 1991), an important component of transcription factor AP-1, thus enhancing its transcriptional activity. In vitro studies revealed that treatment of cells with cytokines, such as TNF and IL-1, as well as exposure to various environmental stresses, including osmotic, redox, and radiation, all resulted in JNK activation (Derijard et al., 1994; Kyriakis et al., 1994; Ip and

Davis, 1998). Phosphorylation of the AP-1 subunits is directly linked to apoptosis

(Karin et al., 1997; Colotta et al., 1992). Apoptotic stimuli by DNA damaging agents ultimately lead to mitochondrial disruption and the release of death-promoting factors, such as cytochrome c. Previous work has demonstrated that apoptosis and mitochondrial damage are controlled in part by the Bcl-2 family of proteins, which can inhibit (i.e., Bcl-2 and Bcl-xL) and or promote (i.e., Bax and Bak) cytochrome c release. Cytochrome c release initiates a self-amplifying cascade of proteolysis among cytosolic caspases that ends in cell death (Roth and Reed, 2002). Early apoptotic events in response to DNA damage lead to the promotion of DNA repair and the activation of poly(ADP-ribose) polymerase-1 (PARP-1) (Soldani and Scovassi, 2002),

154

which transfers ADP-ribose to other nuclear proteins involved in DNA repair and transcription (D'Amours et al., 1999). PARP-1 activation is also thought to be a cell death mediator, although the actual mechanism of PARP-1 induced cell death remains unknown. It has been speculated that utilization of nicotinamide adenine dinucleotide

(NAD+) in PARP-1-initiated ADP-ribosylation leads to depletion of NAD+ stores.

This eventually leads to disrupted mitochondrial function, which stimulates cytochrome c release and caspase activation (Chiarugi and Moskowitz, 2002). With

PARP-1 being caspase substrate, it is targeted for cleavage and inactivated during apoptosis, possibly disrupting PARP-1 activity and subsequent DNA repair, which ultimately allows for the promotion of nuclear disintegration by endonucleases (Chen et al., 2003c). Studies performed by Salas and Burchiel in which Daudi human B cells were treated with B[a]P and B[a]P-7,8-dihydrodiol revealed DNA fragmentation, decreased Bcl-2, and cleavage of PARP-1, further providing evidence that DNA damage may be the leading initiator of apoptosis (Salas and Burchiel, 1998).

Increasing evidence has revealed that arsenic differentially activates these

MAPKs in a variety of human cell lines (Bode and Dong, 2002). Qu and colleagues generated a malignant transformed rat liver TRL1215 cell line by chronic arsenic treatment. Upon challenging the transformed cells with arsenic to monitor changes in apoptosis, they found that the transformed cells were resistant to arsenic-induced apoptosis in comparison to the control. Resistance could be removed by treating cells with a JNK specific activator, indicating arsenic suppression of JNK in transformed cells (Qu et al., 2002). Subsequent studies demonstrated inhibition of JNK activity but increased ERK and p38 activities in these transformed cells. Substantial increases

155

of two anti-apoptotic proteins, Bcl-xL and Bcl-2, and significant decreases in expression of pro-apoptotic protein, Bax, were also observed. These findings show that arsenic treatment disrupts the JNK signaling pathway, which leads to inhibition of apoptosis. However, JNK has also been documented as one of several key stress response protein kinases that is activated in response arsenic (Cavigelli et al., 1996;

Chen et al., 2003a) and is a central mediator of cellular apoptosis (Davis, 2000;

Sabapathy et al., 2001; Kamata et al., 2005) following activation in response to prooxidants. Activation of ROS and JNK in response to toxic challenges can be identified by the subsequent induction of c-jun, c-fos, and junB gene expression

(Cavigelli et al., 1996). Arsenic is a potent inducer of oxidative stress, and has been shown to generate ROS, while inducing JNK1 and JNK2, as well as AP-1 activity. In vitro experiments by Cavigelli et al. demonstrated trivalent arsenic as a potent stimulator of AP-1 transcriptional activity and an efficient inducer of c-fos and c-jun gene expression. Induction of c-jun and c-fos transcription by trivalent arsenic additionally corresponded with increased activation of JNK and p38, which phosphorylate transcription factors that activate these immediate early genes

(Cavigelli et al., 1996). Interestingly, prolonged JNK activation, which promotes apoptosis stimulated by activation of mitochondrial pro-apoptotic proteins has been linked to tissue damage (Guma et al., 2009), obesity (Hirosumi et al., 2002), and cancer (Sakurai et al., 2006), while JNK inhibition prevents mitochondrial apoptosis, oxidative stress, and lowers development of chemical-induced cancers (Shibata et al.,

2008). Therefore, the relationship between JNK and ROS may play an important regulatory role in control of intestinal UGT1A1 gene expression following arsenic

156

exposure. Regardless of the contrasting nature of the results, these studies reflect the ability of arsenic exposure to differentially affect the individual MAPKs to produce opposing effects on cell growth and differentiation (Qian et al., 2003). Since these findings implicate the MAPK signaling pathways as important regulators of homeostasis during times of cellular stress, it is possible that DMEs may be regulated by the MAPKs to help mediate the response to various exposures. With previous evidence indicating that MAPK pathways impact UGT1A1, signaling alterations due to arsenic exposure can therefore potentially have significant effects on UGT gene expression.

Cell Cycle Dysregulation and Morphological Changes Occur with Arsenic Exposure

Exposure to the environmental toxicant arsenic is reported to produce a variety of effects, including disruption of signal transduction pathways, cellular proliferation, and apoptosis. It is therefore possible that arsenite may not have specific targets but instead extremely broad effects. Altered cell cycle regulation and morphological changes may contribute to the observed alterations in gene regulation and expression with arsenic exposure (Yancy et al., 2005).

The events involved with the replication and partition of chromosomes are common to all cell cycles, as newly divided cells must receive a full genome complement to survive. In 1953, with the discovery of DNA’s double helical structure by Watson and Crick, it was noted that the specific pairing within the double helix suggested a possible copying mechanism for genetic material (Watson and Crick,

157

1953). Around the same time, microspectrophotometric (Swift, 1950) and autoradiographic (Howard and Pelc, 1953) studies in eukaryotic cells revealed that

DNA replication happened during a restricted portion of interphase termed the S phase. This work eventually led to the eukaryotic cell cycle being divided G1, S, G2, and M (mitosis) phase (Mitchison, 1971). Multiple signals exist to regulate the onset of cell cycle phases and ensure proper cell growth and tissue homeostasis (Nurse,

2000). In a variety of eukaryotic cells, the orderly progression of dividing cells through the cell cycle is controlled by a series of cell cycle regulatory proteins, mainly cyclins, that exert their function by binding to and activating a number of specific cyclin-dependent kinases (CDKs). CDK activity is further regulated by kinases and phosphatases that phosphorylate and dephosphorylate CDKs, respectively. In addition, several specific CDK inhibitory proteins and cell cycle checkpoint proteins, such as Gadd45, have been identified (Sheikh et al., 2000).

Most cells within a normal tissue may be forced out of active cell cycling and into the quiescent (G0) state, from which they may reenter cell cycling under some future circumstances. In mature tissues, cells may be induced to terminal differentiation by relinquishing their proliferative or cell cycling potential (Chen and

Shi, 2002a). Emerging evidence has demonstrated that a variety of stress inducers, including DNA-damaging agents, can activate checkpoint function of cells, leading to cell cycle arrest. There are several checkpoints, existing in G1/S phase, G2 phase, and

M phase of cell cycle, which have surveillance systems to detect specific DNA structures indicative of damage or ongoing repair and replication (Chen and Shi,

2002a). Microarray analysis, RT-PCR, and immunohistochemistry have shown that

158

arsenic increases expression of cyclin D1 (Chen et al., 2004), a key regulatory protein in cell proliferation and tumor formation (Vogt and Rossman, 2001). Increased expression of cell cycle regulatory genes (cyclin G1, PKC delta) is also observed in normal human epidermal keratinocytes treated with non-toxic doses of As3+ (Hamadeh et al., 2002). Experiments carried out by Jia et al. to investigate the influence of arsenic on expression of cyclin related genes in HL60 cells revealed that 82 genes

(including cyclin B1, PCNA, and insulin-like growth factor binding protein) exhibited changes in expression in response to As2O3 treatment (Jia et al., 2003). It was subsequently hypothesized that cyclin B1, PCNA, and insulin like growth factor binding protein might play a significant role in the induction of apoptosis by As2O3.

In vitro arsenic exposure also caused G2/M arrest in human fibroblast cells, which is evident from the delayed upregulation of cell cycle regulatory genes, such as CCNG1,

CCNF, CDC2, CDC25A, and CDKN2A. Other cell cycle control genes, such as

CCNB1, CLK1, and RAD9, were also enhanced by arsenite treatment. Ultimately, activation of both positive and negative regulators of cell cycle control by arsenite indicates that arsenite treatment causes cell cycle dysregulation (Yih et al., 2002).

Toxic metals, such as arsenic, have also been identified as protein phosphatase inhibitors (Cavigelli et al., 1996). Therefore, with phosphatases required for progression from G1/S phase and G2/M phase, metal-induced inhibition of phosphatase activity can delay cell cycle transitions. In addition, arsenic has been shown to alter cell cycle control, causing G1 and/or G2/M phase arrest with subsequent programmed cell death. The in vitro effect of As2O3 on proliferation, cell cycle regulation, and apoptosis was observed in human myeloma cell lines. As2O3

159

significantly inhibited proliferation in all of the myeloma cell lines via cell cycle arrest in association with induction of p21 and apoptosis (Park et al., 2000). Since p53 plays a guarding role in maintaining genome integrity and accuracy of chromosome segregation, the mechanistic effects of arsenite on p53 activation were analyzed in human fibroblasts. Arsenite-induced DNA strand breaks were confirmed via comet assay and cell cycle retardation, and G2-M arrest was observed in 5-bromo-2'- deoxyuridine (BrdU) pulse-labeled cells by flow cytometry. Significant induction of p53 and its downstream protein p21 (Yih and Lee, 2000), as demonstrated by immunoblotting and immunofluorescence, associated with G1 and G2/M arrest and apoptosis suggests that modulations in cell cycle control by arsenite exposure may impact expression of other cellular components. However, studies by Bonzo et al. demonstrated that while arsenite interrupted cell cycle control by initiating G2/M arrest, arsenite-induced inhibition of AhR-mediated TCDD-inducible expression of

CYP1A1 occurred independent of cell cycle control (Bonzo et al., 2005). Although cell cycle control was previously shown to influence CYP1A1 expression through mechanisms involving AhR and other independent pathways, experiments using a range of arsenite concentrations from subcytotoxic to levels that cause cellular arrest and apoptosis demonstrated that inhibition of CYP1A1 induction occurred at concentrations of arsenite well below those that initiated cell cycle arrest and apoptosis. Investigation of arsenite effects on human CYP1A1 gene expression in primary hepatocytes from transgenic mice in combination with polymerase II recruitment analysis has also provided additional evidence that arsenite inhibits

CYP1A1 expression by modifying transcription independent of cell cycle control

160

(Bonzo et al., 2005).

Morphological alterations in arsenic-exposed cells have implicated underlying disruption of cytoskeletal structural elements responsible for cellular integrity, shape, and locomotion. However, specifics of these resulting structural changes are still not understood (Bernstam and Nriagu, 2000). In vitro studies with sodium arsenite have demonstrated similar cytogenetic alterations in a variety of cell systems (Lee et al.,

1985a; Yih and Lee, 1999; Wang and Huang, 1994; Oya-Ohta et al., 1996). In addition, arsenite-treated Syrian hamster embryo cells exhibited induced cytogenetic changes that were closely associated with induced morphological transformation

(Oshimura and Barrett, 1986; Lee et al., 1985b). Perturbation of spindle dynamics was also observed with arsenite exposure in cultured human cells, which resulted in chromosome malsegregation during mitosis (Yih et al., 1997; Huang and Lee, 1998).

Furthermore, Yancy et al. reported on the effects of sodium arsenite on focal adhesion in H9C2 myoblasts. Sublethal arsenite concentrations decreased cell migration, cell attachment, single cell-spreading area, and distribution and number of focal adhesions.

Phospho-protein detection revealed that arsenite decreased both Tyr phosphorylation of focal adhesion kinase (FAK) as well as its auto-phosphorylation at Tyr397, a typical indicator of FAK activation (Parsons, 2003). Decreased Tyr397 phosphorylation of FAK subsequently led to a reduced phosphorylation of the adhesion-related protein, paxillin. This mechanism is essential for focal adhesion formation and important for signaling events of arsenic-induced toxicity (Yancy et al.,

2005; Liu and Waalkes, 2005). These findings suggest that the genotoxicity of arsenic exposure may be due to its ability to induce cytogenetic alterations and/or genetic

161

instability that can subsequently affect signaling cascades and therefore gene expression patterns (Yih and Lee, 2000).

Increased proliferation of the endoplasmic reticulum (ER) has also been directly linked to the increased expression of DMEs, including the UGTs. In 1976,

Banjo and Nemeth measured the UGT activities and the concentrations of ER in 5- and 11-day chick embryo liver during culture, with and without phenobarbital treatment. It was found that UGT and ER always increased in a constant ratio of 2.2 x

10-9 units of transferase activity per square micrometer of membrane, thus demonstrating that synthesis and degradation of UGTs are coupled with ER synthesis and degradation (Banjo and Nemeth, 1976). It therefore follows that xenobiotics and environmental contaminants that cause proliferation can potentially affect DME content, including the UGTs.

Experimental

Materials. Primers for quantitative real-time polymerase chain reaction (Q-PCR) were commercially synthesized at Integrated DNA Technologies, Inc (IDT, San

Diego, CA). The mouse anti-human UGT1A1 antibody was a gift of Dr. Joseph K.

Ritter (Virginia Commonwealth University, Medical College of Virginia, Richmond,

VA). The mouse anti-PCNA antibody was obtained from BD Biosciences (Franklin

Lakes, New Jersey). The following antibodies were obtained from Cell Signaling

Technology, Inc. (Danvers, MA): rabbit anti-p44/p42 MAPK (Erk1/2), rabbit anti- phospho-p44/42 MAPK (Erk1/2) and rabbit anti-phospho- SAPK/JNK. Mouse anti-

JNK1/2 was purchased from BD Biosciences (Franklin Lakes, New Jersey). The

162

primary rabbit anti-Ki-67 antibody was obtained from Genetex, Inc. (San Antonio,

Texas). The secondary biotin goat anti-rabbit Ig antibody was purchased from BD

Biosciences (Franklin Lakes, New Jersey). The DAB Substrate Kit for peroxidase was purchased from Vector Laboratories (Burlingame, CA). All other chemicals and solvents were of analytical grade or the highest grade commercially available.

Animals and Treatments. Humanized UGT1 mice (Tg(UGT1A1*28)Ugt1-/-) were developed previously in a C57BL/6 background (Fujiwara et al., 2010b). To generate hUGT1/Car−/− mice, hUGT1 mice were crossed with Car-null mice provided to our laboratory by Dr. Masahiko Negishi at the National Institute of Environmental Health

Sciences (Ueda et al., 2002). Car null mice were then crossed into the C57BL/6 background before breeding with hUGT1 mice. IKK-αF/F/IKK-βF/F and Vil-Cre/IKK-

αF/F/IKK-βF/F mice were previously generated in a C57BL/6 background (Chen et al.,

2003b). All animals were housed in plastic cages with hardwood chips for bedding in a 12-hour light, 12-hour dark cycle with water and food (#7912, Harlan-Teklad,

Indianapolis, IN) ad libitum. 10 mg/kg metal contaminant dosages were prepared by dissolving the metal salts in water. The control vehicle was water. 12-day-old mice were treated orally and tissues were collected at 14 days after birth. For tissue collection, mice were anesthetized by isoflurane inhalation and the liver was perfused with ice-cold 1.15% KCl. Then, the small intestine was either prepared for histological analysis or opened, rinsed in cold 1.15% KCl, and stored at -80°C for later use. All animal experiments were carried out following University of California San

Diego Institutional Animal Care and Use guidelines.

163

Bilirubin Measurements. Blood was obtained from the submandibular vein and centrifuged at 2000 x g for 5 min. Serum samples (20 µL) were immediately measured for total serum bilirubin using a Unistat Bilirubinometer (Reichert, Inc.,

Depew, NY).

Q-PCR Analysis. Total RNA from whole tissues was isolated using TRIzol reagents according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). One microgram of RNA was reverse-transcribed into cDNA using the iScript cDNA

Synthesis Kit (BioRad, Hercules, CA). Q-PCR was performed with qPCR MasterMix

Plus for SYBR (Eurogentec, Seraing, Belgium), and the reactions were run in the

Mx4000 Multiplex QPCR System (Stratagene, La Jolla, CA). The forward and reverse primers used were: UGT1A1-S, 5'-CCT TGC CTC AGA ATT CCT TC-3' and

UGT1A1-AS, 5'-ATT GAT CCC AAA GAG AAA ACC AC-3'; mouse Cyp2b10-S,

5’-AAA GTC CCG TGG CAA CTT CC-3’ and Cyp2b10-AS, 5’-CAT CCC AAA

GTC TCT CAT GG-3’; mouse Cyp3a11-S, 5’-CTC AAT GGT GTG TAT ATC CCC-

3’, and Cyp3a11-AS, 5’-CCG ATG TTC TTA GAC ACT GCC-3’; mouse Cyp4a10-

S, 5’-CAT GTT CGA GGG CCA TGA-3’, and Cyp4a10-AS, 5’-TGT GGC CAG

ATA GAA GAT-3’; mouse Gsta1-S, 5’-CAG CCT CCC CAA TGT GAA GAA-3’, and Gsta1-AS, 5’-TGG CTC CAT CAA TGC AGC TT-3’; mouse Gsta2-S, 5’-AGC

TTG ATG CCA GCC TTC TGA-3’, and Gsta2-AS, 5’-TTT CTC TGG CTG CCA

GGA TGT-3’; mouse SOD-1-S, 5’-CGA TGA AAG CGG TGT GCG TGC TG-3’, and SOD-1-AS, 5’-TCT CCA ACA TGC CTC TCT TCA TC-3’; mouse SOD-2-S, 5’-

164

AGA GCA GCG GTC GTG TAA ACC T-3’, and SOD-2-AS, 5’-CCA GAG CCT

CGT GGT ACT TCT C-3’; mouse CAT-S, 5’-ACC AGG GCA TCA AAA ACT TG-

3’, and CAT-AS, 5’-GCC CTG AAG CTT TTT GTC AG-3’; mouse GPx-1-S, 5’-

GGT TCG AGC CCA ATT TTA CA-3’, and GPx-1-AS, 5’-TCG ATG TCG ATG

GTA CGA AA-3’; mouse Nqo1-S, 5’-GGT GAT ATT TCA GTT CCC ATT GC-3’, and Nqo1-AS, 5’-GCA GGA TGC CAC TCT GAA TC-3’; mouse HO-1-S, 5’-CAG

GTG TCC AGA GAA GGC TTT-3’, and HO-1-AS, 5’-TCT TCC AGG GCC GTG

TAG AT -3’; mouse COX-2-S, 5’-GCA GGA TGC CAC TCT GAA TC-3’, and

COX-2-AS, 5’- GCT CGG CTT CCA GTA TTG AG -3’; mouse GSH-Re-S, 5’- GCG

TGA ATG TTG GAT GTG TAC C -3’, and GSH-Re-AS, 5’-TTC CCA TTG ACT

TCC ACC GTG G-3’; mouse Tff1-S, 5’-AAA CAT GTA TCA TGG CCC-3’, and

Tff1-AS, 5’-GAA TTC GAG GAC TAA AAG TCT-3’; mouse Tff2-S, 5’-TGC TTT

GAT CTT GGA TGC TG-3’, and Tff2-AS, 5’-GGA AAA GCA GCA GTT TCG AC-

3’; mouse Tff3-S, 5’-GCT GCC ATG CAG ACC AGA GCC-3’, and Tff3-AS, 5’-

TGG CCA CCA TCA GCA GCA GG-3’; mouse cyclophilin (CPH)-S, 5'-CAG ACG

CCA CTG TCG CTT T-3' and mCPH-AS, 5'-TGT CTT TGG AAC TTT GTC TGC

AA-3'. Each reaction contained 0.75 µL of cDNA and 0.25 µM of the primers in a total volume of 15 µL. PCR conditions were: 95°C for 10 min followed by 40 cycles of 95°C for 15 sec, 60°C for 20 sec, and 72°C for 40 sec.

Western Blot Analysis. Whole cell lysates from whole tissues were prepared with

RIPA Buffer plus protease inhibitors. Briefly, 100 mg of tissue was homogenized on ice in 750 µl of the above buffer. Homogenates were incubated on ice for 20 min. and

165

vortexed occasionally. Homogenates were then centrifuged at 14,000 rpm for 20 min. at 4°C and supernatants were collected for analysis. For phospho-protein detection, cytosolic fractions were prepared. Briefly, 100 mg of tissue was homogenized on ice in 1 mL of RIPA buffer plus protease and phosphatase inhibitors. Nuclei and unbroken cells were removed by low-speed centrifugation (1000 x g for 10 min. at

4°C) and the supernatant was centrifuged at 100,000 x g for 1 hour at 4°C.

Supernatants were used for phospho-western analysis. All Western blots were performed using NuPAGE Bis-Tris polyacrylamide gels as outlined by the supplier

(Invitrogen, Carlsbad, CA). Protein was heated at 70°C for 10 min in loading buffer and resolved in 4-12% Bis-Tris gels under denaturing conditions (50 mM MOPS, 50 mM Tris-base, pH 7.7, 0.1% SDS, 1mM EDTA). The resolved protein was transferred onto nitrocellulose membrane using a semidry transfer system. The membrane was blocked with 5% nonfat dry milk in 10 mM Tris74 HCl, pH 8, 0.15 M

NaCl, and 0.05% Tween 20 (Tris-buffered saline) for 1 hour at room temperature.

The membrane was washed in the Tris-buffered saline solution and incubated with primary antibodies in Tris-buffered saline, shaking at 4°C overnight. Membranes were then washed five times with Tris-buffered saline solution and incubated with horseradish peroxidase-conjugated secondary antibodies in Tris-buffered saline solution with 2% nonfat milk for 1 hour at room temperature. Membranes were washed five times with Tris-buffered saline solution and visualized using chemiluminescent reagents, on the BIORAD ChemiDoc XRS chemiluminescence detection system.

166

Histology. The small intestine was collected 48 hours after treatment, flushed with

10% formalin, and fixed overnight in 10% formalin. Tissues were then transferred to

70% ethanol, embedded in paraffin, and sectioned for slides. Slides were stained with hematoxylin and eosin for H&E or Schiff reagent for Periodic Acid Schiff (PAS) histological analysis. Ki-67 immunostaining was performed using the primary rabbit anti-Ki-67 antibody, secondary biotin goat anti-rabbit Ig antibody, and Strep-HRPO antibody.

Results

Intestinal UGT1A1 Induction Occurs with Oral Arsenic Exposure

The UGT1A1 gene is responsive to a multitude of ligands because of the complex enhancer module distal to the promoter region. Since numerous environmental toxicants can induce UGT1A1 through association with the xenobiotic receptors, it can be speculated that exposure of hUGT1 mice with environmental toxicants can result in reduced serum bilirubin levels through induction of UGT1A1 expression. To evaluate the hUGT1 mice model as a sensor for environmental exposure, we treated mice orally with arsenic (As3+), cadmium (Cd2+), lead (Pb2+), iron (Fe2+), copper (Cu2+), and Chromium (Cr6+) with a dose of 10 mg/kg at 12 days after birth. Compared with the water-treated controls, As3+- and Cd2+-treated mice exhibited significantly lower serum bilirubin levels at 14 days after birth, while Pb2+-,

Fe2+-, Cu2+-, and Cr6+-treated hUGT1 mice still showed higher serum bilirubin levels, relative to the As3+- and Cd2+-treated mice (Figure 3-1A). To determine which tissue was responsible for increased bilirubin metabolism in the As3+- and Cd2+-treated

167

hUGT1 mice, RNA was isolated from liver and small intestine and Q-PCR analysis was performed for UGT1A1. In the liver, which is typically known as the major tissue for bilirubin metabolism, the expression level of UGT1A1 was the same between the control and As3+-treated mice (Figure 3-1B). In contrast, UGT1A1 levels were markedly induced in the small intestine of As3+-treated mice (Figure 3-1C).

CAR is not Involved in Regulating Arsenic-Induced UGT1A1 Expression

Arsenic can activate various transcription factors by triggering an oxidative stress response. It was therefore speculated that the induction of intestinal UGT1A1 by As3+ could be modulated through the activation of certain transcription factors.

The xenobiotic receptors, such as AhR, PPARα, CAR, and PXR are widely known for their involvement in regulating UGT1A1 gene expression by binding specific enhancer sequences within the PBREM region (Sugatani et al., 2005a; Senekeo-Effenberger et al., 2007; Yueh et al., 2003; Xie et al., 2003). To determine if the xenobiotic receptors are responsible for UGT1A1 induction in the small intestine with As3+ exposure, the expression of several xenobiotic receptor target genes was examined, including Cyp1a1 (AhR), Cyp2b10 (CAR), Cyp3a11 (PXR), and Cyp4a10 (PPARα) in control and As3+ treated mice. There was no induction of intestinal Cyp1a1,

Cyp3a11, or Cyp4a10, eliminating involvement of AhR, PXR and PPARα in response to As3+ (Figure 3-2A,C, D). However, there was a statistically significant induction of

Cyp2b10 gene expression (Figure 3-2B), implicating a potential role for CAR in regulating UGT1A1 in the GI tract following As3+ treatment. To directly examine the role of CAR in regulating UGT1A1 gene expression, recently generated hUGT1Car-/-

168

mice were treated with As3+. CAR is non-functional in the hUGT1Car-/- mouse model, such that potent CAR agonist, PB, cannot induce UGT1A1 and Cyp2b10 in the mice. However, it was observed that UGT1A1 expression was still induced and serum bilirubin levels decreased with As3+ treatment in neonatal hUGT1Car-/- mice (Figure

3-3A,B). In addition, while the CAR target gene, Cyp2b10, was not induced with PB treatment, its expression was still upregulated with As3+ treatment in neonatal hUGT1Car-/- mice. This indicates that induction of UGT1A1 and Cyp2b10 with As3+ exposure in hUGT1 mice occurs independent of CAR.

169

Figure 3-1. Effects of metal contaminant exposure on serum bilirubin levels and UGT1A1 expression in neonatal hUGT1 mice. At 12 days after birth, neonatal hUGT1 mice were treated orally with 10 mg/kg of the indicated metal contaminants: sodium arsenite (As3+), cadmium chloride (Cd2+), lead (II) nitrate (Pb2+), iron (II) sulfate (Fe2+), cupric (II) chloride (Cu2+), and potassium chromate (Cr6+). Forty-eight hours post treatment, blood was collected and serum samples were prepared. (A) Serum bilirubin levels were measured using a Bilirubinometer. RNA was isolated from the liver and small intestine and Q-PCR was performed to determine UGT1A1 expression (B) in the liver (C) and small intestine. Data is shown as fold induction of the value observed in mice treated with water alone (CT). Data is expressed as mean ± s.d., n = 6. * P < 0.01.

170

Figure 3-2. Arsenic exposure induces Cyp2b10 in the small intestine. Forty-eight hours after oral As3+ exposure at 12 days, RNA was isolated from the small intestine at 14 days and Q-PCR was performed for Cyp1a1, Cyp2b10, Cyp3a11, and Cyp4a10, target genes of xenobiotic receptors AhR, PXR, CAR, and PPARα, respectively. Data is shown as fold induction of the value observed in mice treated with water alone (CT). Data is expressed as mean ± s.d., n = 3. * P < 0.05.

Figure 3-3. Arsenic exposure induces UGT1A1 and decreases serum bilirubin levels in hUGT1Car-/- mice. At 12 days after birth, neonatal hUGT1Car-/- mice were treated orally with 10 mg/kg As3+. At 14 days and 48 hours after treatment, (A) serum bilirubin levels were quantified and (B) intestinal UGT1A1 levels determined by Q- PCR analysis. (C) Cyp2b10 expression in the small intestine was also determined. Data is shown as fold induction of the value observed in mice treated with water alone (CT). Data is expressed as mean ± s.d., n = 6. * P < 0.01.

171

The NF-κB/IKK Pathway is not Involved in Regulating Intestinal UGT1A1

NF-κB prevents the generation of ROS in response to toxic challenges. In addition, previous work in our lab has demonstrated the existence of a close relationship between NF-κB expression in intestinal cells and regulating the expression of UGT1A1 post cadmium exposure (Fujiwara et al., 2012). These findings led to investigation of the involvement of the NF-κB pathway in modulating

As3+-induced UGT1A1 expression. As3+ has been shown to exert its biological effects by reacting with IKK’s free thiol to inhibit NF-κB signaling (Roussel and

Barchowsky, 2000), as blocked IKK results in limited degradation of IκB and decreased NF-κB activation (Kapahi et al., 2000). Western Blot analysis with small intestinal whole cell lysates revealed increased IκB protein in neonatal hUGT1 mice exposed to As3+ (Figure 3-4A), initially suggesting UGT1A1 induction may result from

NF-κB repression. Further examination of the role of NF-κB/IKK signaling in the induction of gastrointestinal UGT1A1 with As3+ exposure was done using mice in which NF-κB signaling through IκB-kinase (IKK)-α and IKK-β has been selectively ablated in the intestinal epithelium through the conditional knockout of the genes. To perform this experiment, transgenic mice expressing a villin-promoter driven Cre recombinase gene (Vil-Cre) only in intestinal epithelium were crossed with floxed

IKK-αF/F/IKK-βF/F mice. Twelve-day-old IKK-αF/F/IKK-βF/F and Vil-Cre/IKK-

αF/F/IKK-βF/F mice were then treated with 10 mg/kg As3+ and Cyp2b10 expression in the intestinal epithelial cells was determined by Q-PCR. As3+ induced Cyp2b10 in the intestinal epithelial cells of both the control IKK-αF/F/IKK-βF/F mice and Vil-Cre/IKK-

αF/F/IKK-βF/F mice (Figure 3-4B). Although these mice do not carry the UGT1

172

transgene, evaluation of Cyp2b10 expression is still possible, since regulation occurs in a similar fashion to that of human UGT1A1 in hUGT1 mice. These findings suggest that induction of intestinal Cyp2b10 gene expression by As3+ is independent of NF-

κB/IKK signaling.

Figure 3-4. Effects of Arsenic exposure in IKK-α/IKK-β conditional knockout mice. (A) 12 days after birth, neonatal hUGT1 mice were treated orally with 10 mg/kg As3+. At 14 days and 48 hours after treatment, small intestine was collected and whole cell lysates were prepared for Western Blot analysis with the IκB antibody. (B) At 12 days after birth, neonatal IKK-αF/F/IKK-βF/F and Vil-Cre/IKK-αF/F/IKK-βF/F mice were treated orally with 10 mg/kg sodium arsenite. At 14 days after birth and 48 hours after the treatment, Cyp2b10 levels in the intestinal epithelial cells were determined by Q-PCR. Data is shown as fold induction of the value observed in mice treated with water alone (CT). Data is expressed as mean ± s.d., n = 3. * P < 0.01.

Oxidative Stress Induced Nrf2 Activation Upregulates UGT1A1 Expression

As3+ has been identified as a potent inducer of oxidative stress and has been shown to generate ROS, which can lead to activation of signaling pathways involved

173

in regulating genes involved in cellular defense mechanisms. Upregulation of cytoprotective genes, including DMEs, in response to environmental insults is known as the antioxidant response (Yueh and Tukey, 2007). To investigate whether induction of UGT1A1 with As3+ exposure is upregulated as part of the antioxidant response, expression levels of several oxidative stress related genes were quantified.

Two classic Nrf2 genes, Gsta1 and Gsta2, were significantly upregulated in small intestine post exposure (Figure 3-5F,G). ROS can disrupt the Nrf2-Keap1 complex, leading to transcriptional activation of Nrf2 target genes, such as UGT1A1 and the

GSTs (Lee et al., 2005). The involvement of the Nrf2-Keap1 signaling pathway was further investigated by performing co-treatment experiments with the potent antioxidant, NAC, to prevent ROS generation. Mice pre-treated with NAC 1 hour prior to As3+ exposure exhibit significant reductions in UGT1A1, Cyp2b10, and Gsta1 expression (Figure 3-6). NAC pre-treatment caused UGT1A1 induction to decrease by

98%, Cyp2b10 by 77%, and Gsta1 by 98%. These findings implicate that induction may occur through oxidative stress-induced Nrf2 activation.

174

Figure 3-5. Arsenic exposure also induces Gsta1 and Gsta2 in the small intestine. Forty-eight hours after oral As3+ exposure, RNA was isolated from small intestine and Q-PCR was carried out for several oxidative stress related genes. Data is shown as fold induction of the value observed in mice treated with water alone (CT). Data is expressed as mean ± s.d., n = 3. * P < 0.01.

Figure 3-6. Pre-treatment with NAC significantly reduces UGT1A1, Cyp2b10, and Gsta1 expression in small intestine. Mice were pre-treated with NAC 1 hour prior to As3+ exposure. Forty-eight hours after oral As3+ exposure, RNA was isolated from small intestine and Q-PCR was carried out for UGT1A1, Cyp2b10 and Gsta1. Data is shown as fold induction of the value observed in mice treated with water alone (CT). Data is expressed as mean ± s.d., n = 3. * P < 0.05.

175

UGT1A1 Induction Occurs Independent of MAPK Activation

Recent studies have found that induction of ARE-dependent Phase II detoxifying enzymes is mediated by a MAPK pathway (Yu et al., 2000; Keum et al.,

2003; Shen et al., 2004; Yu et al., 1999), and therefore UGT1A1 induction may be occurring through MAPK activation of Nrf2. Changes in protein and phospho-protein expression of two MAPKs in small intestinal cytosolic fractions were used to assess whether oxidative stress induced in intestinal tissue of neonatal hUGT1 mice affected

MAPK activation that could lead to downstream changes in UGT1A1 gene expression.

As3+ exposure did not increase phosphorylation of either JNK or ERK MAPKs (Figure

3-7), indicting that these signaling pathways are not likely involved in transcriptional activation of UGT1A1.

Figure 3-7. Arsenic exposure does not activate JNK or ERK in intestine. Forty- eight hours after oral As3+ exposure, cytosolic fractions were isolated from small intestine and prepared for Western Blot analysis with total JNK1/2, phospho- SAPK/JNK, total ERK1/2, and phospho-ERK1/2 antibodies. CT indicates mice treated with water alone.

176

Arsenic Exposure Causes Intestinal Damage, Changes in Cellular Morphology, and Increases Proliferation

The contribution of As3+ to such a variety of different diseases indicates that

As3+ might not function through one specific mechanism, but instead by eliciting a more global effect. Both abnormal cell cycle regulation (Bonzo et al., 2005; Lau et al., 2004; Eguchi et al., 2011) and changes in cellular morphology due to As3+- induced cytotoxicity (Yancy et al., 2005; Li et al., 2011) have been observed with exposure and can lead to changes in signaling that ultimately affect gene expression.

This directed initial experimental efforts toward the assessment of intestinal damage post exposure. The trefoil factor family (TFF) peptides are involved in maintaining the integrity of the gastrointestinal mucosa (Ribieras et al., 1998), but can be ectopically expressed in cells of regenerating tissue surrounding compromised areas, therefore implicating their protective role in mucosal injury (Playford et al., 1996). To determine if intestinal damage occurs with As3+ exposure, I measured changes in gene expression of Tff1, Tff2, and Tff3 in control and As3+-treated hUGT1 intestinal tissue.

Tff1, which is typically expressed in stomach, is significantly upregulated in the small intestine (Figure 3-8A), while Tff2 and Tff3 expression decreases or remains unchanged, respectively (Figure 3-8B,C). Abnormal induction of Tff1 in the small intestine confirms intestinal injury.

177

Figure 3-8. Tff1 is ectopically induced in small intestine with arsenic exposure. Forty-eight hours after oral As3+ exposure, small intestine was collected and RNA isolated for Q-PCR analysis of Tff1, Tff2, and Tff3. Data is shown as fold induction of the value observed in mice treated with water alone (CT). Data is expressed as mean ± s.d., n = 3. * P < 0.05.

Significant induction of small intestinal Tff1 led to immunohistochemical assessment of intestinal damage. H&E and PAS stainings of the small intestine were performed to assess any obvious morphological differences between control and As3+- treated samples. H&E stained samples exhibit clear differences in goblet cell mucin secretion (Figure 3-9A,B), which is indicative of intestinal damage. However, abnormal expansion or vacuole formation at villi tips was also seen in As3+-treated samples (indicated by black arrows in Figure 3-9B). PAS staining was subsequently used to confirm if the goblet cells and vacuoles were secreting glycoproteins, such as mucin, which is a sign of intestinal damage and part of the normal repair process.

PAS staining revealed increased goblet cell mucin secretion in As3+-treated samples, compared to the controls (mucin stains bright magenta in Figure 3-10A,B). However, an absence of PAS staining within the abnormal vacuoles formed at the villi tips suggests that this is not a normal maintenance response (indicated by black arrows in

Figure 3-10B). Interestingly, PAS staining revealed that As3+-treated samples

178

exhibited stacked and enlarged nuclei (Figure 3-10B), which is indicative of increased cellular proliferation.

Arsenic is known to elicit profound and differential effects on cellular proliferation and apoptosis. In addition, increases in proliferation have been directly linked to increases in UGTs (Banjo and Nemeth, 1976). As such, Proliferating Cell

Nuclear Antigen (PCNA) western blotting and Ki-67 immunostaining were used to investigate changes in cellular proliferation with As3+ exposure. PCNA is only expressed within the nuclei of cells during the DNA synthesis phase, while the Ki-67 antigen expression only occurs during late G1, S, G2, and M phases of the cell cycle and cannot be detected in cells in G0 phase. Increases in both PCNA concentrations

(Figure 3-11A) and Ki-67 immunostaining (Figure 3-11B) were observed in As3+- treated samples. Active proliferation can be seen within the crypts, but there is significantly greater epithelial migration in As3+-treated samples, confirming increased cellular proliferation (Figure 3-11B).

179

Figure 3-9. H&E staining reveals abnormal vacuole formation. The small intestine was collected 48 hours after As3+ treatment, flushed with 10% formalin, and fixed overnight in 10% formalin. Tissues were then transferred to 70% ethanol, embedded in paraffin, and sectioned for slides. Slides were stained with hematoxylin and eosin for H&E histological analysis.

Figure 3-10. PAS staining confirms abnormal vacuole formation. The small intestine was collected 48 hours after As3+ treatment, flushed with 10% formalin, and fixed overnight in 10% formalin. Tissues were then transferred to 70% ethanol, embedded in paraffin, and sectioned for slides. Slides were stained with Schiff reagent for Periodic Acid Schiff (PAS) histological analysis.

180

Figure 3-11. Increased PCNA concentrations and Ki-67 immunostaining confirm increased proliferation. (A) Forty-eight hours after oral As3+ exposure, whole cell lysates were isolated from small intestine and prepared for Western Blot analysis with the PCNA antibody. (B) The small intestine was collected 48 hours after treatment, flushed with 10% formalin, and fixed overnight in 10% formalin. Tissues were then transferred to 70% ethanol, embedded in paraffin, and sectioned for slides. Ki-67 immunostaining was performed using the primary rabbit anti-Ki-67 antibody, secondary biotin goat anti-rabbit Ig antibody and Strep-HRPO antibody.

Discussion

Much of our knowledge about the effects of As3+ has been inferred from indirect epidemiological and clinical observations, especially since As3+ has never been shown conclusively to be an initiating or a promoting agent of carcinogenesis in animals (Bode and Dong, 2002). Therefore, elucidation and understanding of the molecular mechanisms that determine the effects of As3+ exposure are critical for

181

understanding its enigmatic nature. There is particularly little information about

As3+’s ability to modulate expression of DMEs, either directly or indirectly. The generation of a humanized UGT1 mouse model, which expresses the entire human

UGT1 locus in a mouse Ugt1 null background and exhibits elevated serum bilirubin levels during development (Fujiwara et al., 2010b), has enabled studies that probe at the ability of prominent metal contaminants, including As3+, to regulate the human

UGT genes in a relevant in vivo model.

Since the UGT1A1 gene contains a series of xenobiotic receptor enhancer sequences, it is one of the few genes that can be independently modulated by activation of numerous transcriptional factors. Therefore, it can be speculated that exposure of hUGT1 mice to environmental toxicants can reduce serum bilirubin levels through induction of UGT1A1 expression. Oral As3+ exposure in hUGT1 mice caused marked decreases in serum bilirubin that corresponded with significant increases in intestinal UGT1A1. An absence of induction of UGT1A1 was noted in liver, implicating induction as intestinal specific.

In addition to findings supporting the complex and extensive regulation of the

UGT1 locus by various nuclear receptors, studies demonstrating that As3+ is capable of affecting CYP gene expression by affecting AhR and PXR activation (Wu et al., 2009;

Noreault et al., 2005) led to investigating nuclear receptor involvement in the regulation of As3+-induced UGT1A1 expression. Examination of the expression of several xenobiotic receptor target genes in control and As3+ treated mice eliminated involvement of AhR, PXR, and PPARα in response to As3+. However, there was a statistically significant induction of Cyp2b10 gene expression, which implicated a

182

potential role for CAR in regulating UGT1A1 in the GI tract following As3+ treatment. hUGT1Car-/- were utilized to directly examine the role of CAR in regulating UGT1A1 gene expression. Serum bilirubin levels decreased and UGT1A1 and Cyp2b10 were still inducible with As3+ treatment in neonatal hUGT1Car-/- mice, indicating that induction of UGT1A1 and Cyp2b10 with As3+ exposure in hUGT1 mice is not directly regulated by CAR.

Oxidative stress is currently the most widely accepted and studied mechanism of As3+ toxicity (Ercal et al., 2001). As3+ causes oxidative stress and the generation of

ROS, which in turn, activates signaling pathways involved in the regulation of genes encoding antioxidative response enzymes (Pi et al., 2003). Upregulation of cytoprotective genes, including DMEs, in response to environmental insults is known as the antioxidant response. There are many pathways that are affected by ROS, including the NF-κB/IKK pathway, the Nrf2-Keap1 pathway, and the MAPK signaling pathways (Felix et al., 2005; Yueh and Tukey, 2007; Bode and Dong, 2000).

The NF-κB/IKK pathway has been shown to regulate transcription of important protective genes (Chen et al., 2001). As such, changes in its transcriptional activation can alter target gene expression. Arsenic is believed to exert its biological effects by reacting with IKK’s free thiol to block NF-κB signaling. Blocked IKK results in decreased NF-κB translocation and limited degradation of IκB (Kapahi et al., 2000). Increased IκB concentrations were observed in small intestinal whole cell lysates, suggesting NF-κB repression was involved in UGT1A1 induction. However, previous work in our lab demonstrated that hUGT1 mice treated orally with known

NF-κB activators, such as Cd2+ and LPS, (Hyun et al., 2007; Luo et al., 2004),

183

exhibited decreases in serum bilirubin and significant increases in intestinal UGT1A1 and Cyp2b10, which are patterns identical to what is observed in As3+-treated hUGT1 mice. Fujiwara et al. further investigated the role of intestinal NF-κB in Cd2+-induced expression of Cyp2b10 in a conditional knockout mouse model deficient in Ikkα/β specifically in intestinal epithelial cells (Fujiwara et al., 2012). Although these mice do not carry the UGT1 transgene, evaluation of Cyp2b10 expression was still possible, since regulation occurs in a similar fashion to that of human UGT1A1 in hUGT1 mice.

Cyp2b10 induction was completely abolished in Vil-Cre/IkkαF/FIkkβF/F (GI knockout) mice treated with Cd2+, compared to the Cd2+-treated control IkkαF/F/IkkβF/F (WT mice) (Fujiwara et al., 2012). Subsequent investigation into the role of the NF-

κB/IKK pathway in regulating intestinal UGT1A1 expression post As3+ exposure in twelve-day-old IKK-αF/F/IKK-βF/F and Vil-Cre/IKK-αF/F/IKK-βF/F mice revealed sustained induction of Cyp2b10 in the intestinal epithelial cells of both control IKK-

αF/F/IKK-βF/F mice and Vil-Cre/IKK-αF/F/IKK-βF/F mice. While these results suggest that induction of intestinal Cyp2b10 gene expression by As3+ occurs independent of

NF-κB/IKK signaling, it is difficult to conclusively rule out NF-κB involvement in

UGT1A1 gene regulation without repeating As3+ treatments in a hUGT1/IKK-null model in which it is possible to directly measure UGT1A1 mRNA and protein expression.

UGT1A1 has been identified as an Nrf2 target gene that is induced in response to oxidative stress (Yueh and Tukey, 2007). To investigate whether induction of

UGT1A1 by As3+ exposure is upregulated as part of the Nrf2 antioxidant response, the expression levels of several oxidative stress related genes were quantified and it was

184

established that two classic Nrf2 genes, Gsta1 and Gsta2, were significantly upregulated in small intestine post exposure (Lee et al., 2005). NAC pre-treatment of hUGT1 mice to prevent ROS generation subsequently revealed that pre-treatment caused UGT1A1 induction to decrease by 98%, Cyp2b10 by 77%, and Gsta1 by 98%.

These findings implicate that induction may occur through oxidative stress-induced

Nrf2 activation. As previous studies with Nrf2 deficient mouse have highlighted the crucial importance of elevated Phase II gene expression in cytoprotection, it is necessary to confirm Nrf2 involvement in regulating UGT1A1 by treatment experiments in similar model. However, increasing evidence has revealed that As3+ differentially activates MAPKs (Bode and Dong, 2002), which can impact regulation of downstream target genes. Recent studies have found that induction of ARE- dependent Phase II detoxifying enzymes is mediated by a MAPK pathway (Yu et al.,

2000; Keum et al., 2003; Shen et al., 2004; Yu et al., 1999), and therefore UGT1A1 induction may be occurring through MAPK activation of Nrf2. Assessment of protein and phospho-protein of two MAPKs in small intestinal cytosolic fractions revealed that As3+ exposure did not increase phosphorylation of either JNK or ERK MAPKs, thus indicating that these signaling pathways are not likely involved in transcriptional activation of UGT1A1.

The contribution of As3+ to such a variety of different diseases indicates that it might not function through one, specific mechanism, but instead by eliciting a more global effect. Both abnormal cell cycle regulation (Bonzo et al., 2005; Lau et al.,

2004; Eguchi et al., 2011) and changes in cellular morphology due to As3+-induced cytotoxicity (Yancy et al., 2005; Li et al., 2011) have been observed with exposure

185

and can lead to changes in signaling that can affect gene expression. Initial assessment of intestinal damage by As3+ exposure was provided by quantifying changes in gene expression of the Tffs, which are involved in maintaining the integrity of the gastrointestinal mucosa (Ribieras et al., 1998; Playford et al., 1996). Abnormal induction of Tff1 in small intestine, which is typically expressed in stomach suggested intestinal injury, which led to further investigation of damage by immunohistochemical analysis. H&E and PAS stainings of the small intestine were performed to assess any obvious morphological differences between control and As3+- treated samples. While H&E stained samples showed clear differences in goblet cell mucin secretion, abnormal expansion or vacuole formation at villi tips was also seen in As3+-treated samples. PAS staining was subsequently used to confirm if the goblet cells and vacuoles were secreting glycoproteins, such as mucin, which is a sign of intestinal damage and part of the normal repair process. PAS staining revealed increased goblet cell mucin secretion in As3+-treated samples, compared to the controls. Interestingly, the absence of PAS staining within the abnormal vacuoles formed at the villi tips suggests that this is not a normal maintenance response.

Furthermore, PAS staining revealed that As3+-treated samples exhibited stacked and enlarged nuclei that are indicative of increased cellular proliferation. These findings were particularly significant, as both NF-κB and the MAPKs, which are classically involved in regulating cell growth and apoptosis, have been largely ruled out as potential regulators of As3+-induced UGT1A1 expression. Nonetheless, As3+ is known to elicit profound and differential affects on cellular proliferation and apoptosis (Qian et al., 2003). Increases in both PCNA concentrations and Ki-67 immunostaining were

186

observed in As3+-treated samples. Active proliferation can be seen within the crypts, but there is significantly greater epithelial migration in As3+-treated samples, ultimately confirming increased cellular proliferation.

Increases in proliferation have been directly linked to increases in UGT content

(Banjo and Nemeth, 1976). It is very possible that proliferation could ultimately be underlying the induction that is observed with As3+ exposure. The overall contribution of proliferation can be further assessed in hUGT1/growth arrest and DNA-damage- inducible gene 45β (Gadd45β) deficient mice. The three Gadd45 genes (Gadd45α,

Gadd45β, and Gadd45γ) are all inducible by various environmental stresses, such as

UV and γ-irradiation and oxidative stress. However, unlike the two other homologs,

Gadd45β plays an anti-apoptotic role (Columbano et al., 2005) and therefore Gadd45β null mice would exhibit decreased proliferation. Previous works showing the inducibility of the Gadd45 family by both partial hepatectomy and by treatment with

TCPOBOP, a CAR activator that also produces a particularly strong and rapid proliferative response in mouse liver, have also linked hepatocyte proliferation by

Gadd45β to CAR activation (Tian et al., 2011; Columbano et al., 2005; Costa et al.,

2005). Most recently, work from the Negishi lab has showed that CAR induction by

PB results in the promotion of hepatocellular carcinoma (HCC) through Gadd45β and that the development decreased in Car -/- mice, thus confirming Gadd45β as a CAR target gene (Yamamoto et al., 2010). The work in this dissertation has shown that

UGT1A1 remains inducible in hUGT1/Car -/- mice. Therefore, if histological examination reveals decreases in proliferation, it can follow that induction occurs through a different mechanism.

187

Figure 3-12. The proposed mechanism for oral arsenic-induced UGT1A1 gene expression.

In the process of elucidating the mechanism by which As3+ modulates intestinal UGT1A1, I have narrowed my findings down to three possible mechanisms: the Nrf2-Keap1 pathway, the NF-κB/IKK pathway, and proliferation (Figure 3-12).

Since UGT1A1, Cyp2b10, and Gsta1 expression decreased significantly with NAC pre-treatment, it is reasonable to assume that ROS induced by As3+ activates Nrf2 and in turn upregulates UGT1A1. Maintained induction of Cyp2b10 in the IKK-/- mouse model indirectly suggested that UGT1A1 induct is not mediated through NF-κB.

However, previous experiments in our lab have demonstrated that typical NF-κB inducers, like Cd2+ upregulate UGT1A1 and Cyp2b10 via the NF-κB/IKK pathway in hUGT1 mice. Lastly, proliferation could ultimately be underlying the induction we see. Although direct regulation of UGT1A1 by CAR has been negated, a proliferative response that results in increased UGT1A1 could be indirectly modulated by CAR.

Figure 3-12 summarizes this proposed mechanism. These findings have provided

188

advancements in understanding how As3+ regulates UGT1A1 gene expression and highlight the enigmatic and complicated nature of its exposure. The shocking and on- going prevalence of contamination throughout the world is exactly why it is essential to understand how As3+ can affect important biological processes, such as drug metabolism.

Chapter 3, in part, is currently being prepared for submission for publication of the material. I was the primary investigator and author of this material.

References

Adler V, Polotskaya A, Wagner F and Kraft AS (1992) Affinity-purified c-Jun amino- terminal protein kinase requires serine/threonine phosphorylation for activity. The Journal of biological chemistry 267:17001-17005.

Anderson MT, Staal FJ, Gitler C, Herzenberg LA and Herzenberg LA (1994) Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America 91:11527-11531.

Baldwin AS, Jr. (2001) Series introduction: the transcription factor NF-kappaB and human disease. The Journal of clinical investigation 107:3-6.

Banjo AO and Nemeth AM (1976) Proliferation of endoplasmic reticulum with its enzyme, UDP-glucuronyltransferase, in chick embryo liver during culture. Effects of phenobarbital. The Journal of cell biology 70:319-325.

Barchowsky A, Dudek EJ, Treadwell MD and Wetterhahn KE (1996) Arsenic induces oxidant stress and NF-kappa B activation in cultured aortic endothelial cells. Free radical biology & medicine 21:783-790.

Barchowsky A, Klei LR, Dudek EJ, Swartz HM and James PE (1999) Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite. Free radical biology & medicine 27:1405-1412.

Bernstam L and Nriagu J (2000) Molecular aspects of arsenic stress. Journal of toxicology and environmental health Part B, Critical reviews 3:293-322.

189

Blumberg B, Sabbagh W, Jr., Juguilon H, Bolado J, Jr., van Meter CM, Ong ES and Evans RM (1998) SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes & development 12:3195-3205.

Bode A and Dong Z (2000) Apoptosis induction by arsenic: mechanisms of action and possible clinical applications for treating therapy-resistant cancers. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy 3:21-29.

Bode AM and Dong Z (2002) The paradox of arsenic: molecular mechanisms of cell transformation and chemotherapeutic effects. Critical reviews in oncology/hematology 42:5-24.

Bonzo JA, Belanger A and Tukey RH (2007) The role of chrysin and the ah receptor in induction of the human UGT1A1 gene in vitro and in transgenic UGT1 mice. Hepatology 45:349-360.

Burchiel SW, Davis DA, Ray SD and Barton SL (1993) DMBA induces programmed cell death (apoptosis) in the A20.1 murine B cell lymphoma. Fundamental and applied toxicology : official journal of the Society of Toxicology 21:120-124.

Buzard GS and Kasprzak KS (2000) Possible roles of nitric oxide and redox cell signaling in metal-induced toxicity and carcinogenesis: a review. Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer 19:179-199.

Carroll L (2012) High arsenic levels found in organic foods, baby formula, NBC News Health, NBCNews.com.

Cavigelli M, Li WW, Lin A, Su B, Yoshioka K and Karin M (1996) The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. The EMBO journal 15:6269-6279.

Cerutti PA (1985) Prooxidant states and tumor promotion. Science 227:375-381.

Chen CJ, Chen CW, Wu MM and Kuo TL (1992) Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. British journal of cancer 66:888-892.

Chen F, Castranova V, Li Z, Karin M and Shi X (2003a) Inhibitor of nuclear factor kappaB kinase deficiency enhances oxidative stress and prolongs c-Jun NH2-terminal kinase activation induced by arsenic. Cancer research 63:7689-7693.

190

Chen F, Ding M, Castranova V and Shi X (2001) Carcinogenic metals and NF-kappaB activation. Molecular and cellular biochemistry 222:159-171.

Chen F and Shi X (2002a) Intracellular signal transduction of cells in response to carcinogenic metals. Critical reviews in oncology/hematology 42:105-121.

Chen F and Shi X (2002b) Signaling from toxic metals to NF-kappaB and beyond: not just a matter of reactive oxygen species. Environmental health perspectives 110 Suppl 5:807-811.

Chen H, Li S, Liu J, Diwan BA, Barrett JC and Waalkes MP (2004) Chronic inorganic arsenic exposure induces hepatic global and individual gene hypomethylation: implications for arsenic hepatocarcinogenesis. Carcinogenesis 25:1779-1786.

Chen LW, Egan L, Li ZW, Greten FR, Kagnoff MF and Karin M (2003b) The two faces of IKK and NF-kappaB inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia-reperfusion. Nature medicine 9:575-581.

Chen S, Nguyen N, Tamura K, Karin M and Tukey RH (2003c) The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8- dihydrodiol-9,10-epoxide-induced apoptosis. The Journal of biological chemistry 278:19526-19533.

Chiarugi A and Moskowitz MA (2002) Cell biology. PARP-1--a perpetrator of apoptotic cell death? Science 297:200-201.

Chin BY, Choi ME, Burdick MD, Strieter RM, Risby TH and Choi AM (1998) Induction of apoptosis by particulate matter: role of TNF-alpha and MAPK. The American journal of physiology 275:L942-949.

Cohen E (2011) Report: Arsenic in apple and grape juice, CNN Health, CNN.com.

Colotta F, Polentarutti N, Sironi M and Mantovani A (1992) Expression and involvement of c-fos and c-jun protooncogenes in programmed cell death induced by growth factor deprivation in lymphoid cell lines. The Journal of biological chemistry 267:18278-18283.

Columbano A, Ledda-Columbano GM, Pibiri M, Cossu C, Menegazzi M, Moore DD, Huang W, Tian J and Locker J (2005) Gadd45beta is induced through a CAR- dependent, TNF-independent pathway in murine liver hyperplasia. Hepatology 42:1118-1126.

191

Corsini E, Asti L, Viviani B, Marinovich M and Galli CL (1999) Sodium arsenate induces overproduction of interleukin-1alpha in murine keratinocytes: role of mitochondria. The Journal of investigative dermatology 113:760-765.

Costa RH, Kalinichenko VV, Tan Y and Wang IC (2005) The CAR nuclear receptor and hepatocyte proliferation. Hepatology 42:1004-1008.

D'Amours D, Desnoyers S, D'Silva I and Poirier GG (1999) Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. The Biochemical journal 342 ( Pt 2):249-268.

Davis RJ (2000) Signal transduction by the JNK group of MAP kinases. Cell 103:239- 252.

Ercal N, Gurer-Orhan H and Aykin-Burns N (2001) Toxic metals and oxidative stress part I: mechanisms involved in metal-induced oxidative damage. Current topics in medicinal chemistry 1:529-539.

Evans CD, LaDow K, Schumann BL, Savage RE, Jr., Caruso J, Vonderheide A, Succop P and Talaska G (2004) Effect of arsenic on benzo[a]pyrene DNA adduct levels in mouse skin and lung. Carcinogenesis 25:493-497.

Fairbrother A (2012) Pfizer's Fight to Keep Arsenic in Chicken Feed, The Washington Spectator, The Washington Spectator.

Falkner KC, Pinaire JA, Xiao GH, Geoghegan TE and Prough RA (2001) Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: involvement of both the glucocorticoid and pregnane X receptors. Molecular pharmacology 60:611-619.

Felix K, Manna SK, Wise K, Barr J and Ramesh GT (2005) Low levels of arsenite activates nuclear factor-kappaB and activator protein-1 in immortalized mesencephalic cells. Journal of biochemical and molecular toxicology 19:67-77.

Flora SJ (2011) Arsenic-induced oxidative stress and its reversibility. Free radical biology & medicine 51:257-281.

192

Fry RC, Navasumrit P, Valiathan C, Svensson JP, Hogan BJ, Luo M, Bhattacharya S, Kandjanapa K, Soontararuks S, Nookabkaew S, Mahidol C, Ruchirawat M and Samson LD (2007) Activation of inflammation/NF-kappaB signaling in infants born to arsenic-exposed mothers. PLoS genetics 3:e207.

Fujiwara R, Nguyen N, Chen S and Tukey RH (2010) Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus. Proceedings of the National Academy of Sciences of the United States of America 107:5024-5029.

Garber L (2012) Levels of Arsenic in Rice Skyrocket, FDA Urged to Set Standards Natural Society, Natural Society.

Garcia-Chavez E, Santamaria A, Diaz-Barriga F, Mandeville P, Juarez BI and Jimenez-Capdeville ME (2003) Arsenite-induced formation of hydroxyl radical in the striatum of awake rats. Brain research 976:82-89.

Goodwin B, Hodgson E, D'Costa DJ, Robertson GR and Liddle C (2002a) Transcriptional regulation of the human CYP3A4 gene by the constitutive androstane receptor. Molecular pharmacology 62:359-365.

Goodwin B, Redinbo MR and Kliewer SA (2002b) Regulation of cyp3a gene transcription by the pregnane x receptor. Annual review of pharmacology and toxicology 42:1-23.

Grush L (2012) Arsenic in drinking water deemed 'safe' could harm mothers and children, study finds, Fox News Health, FoxNews.com.

Guma M, Rius J, Duong-Polk KX, Haddad GG, Lindsey JD and Karin M (2009) Genetic and pharmacological inhibition of JNK ameliorates hypoxia-induced retinopathy through interference with VEGF expression. Proceedings of the National Academy of Sciences of the United States of America 106:8760-8765.

Hamadeh HK, Trouba KJ, Amin RP, Afshari CA and Germolec D (2002) Coordination of altered DNA repair and damage pathways in arsenite-exposed keratinocytes. Toxicological sciences : an official journal of the Society of Toxicology 69:306-316.

He X, Chen MG, Lin GX and Ma Q (2006) Arsenic induces NAD(P)H-quinone oxidoreductase I by disrupting the Nrf2 x Keap1 x Cul3 complex and recruiting Nrf2 x Maf to the antioxidant response element enhancer. The Journal of biological chemistry 281:23620-23631.

193

He X and Ma Q (2010) Critical cysteine residues of Kelch-like ECH-associated protein 1 in arsenic sensing and suppression of nuclear factor erythroid 2-related factor 2. The Journal of pharmacology and experimental therapeutics 332:66-75.

Hibi M, Lin A, Smeal T, Minden A and Karin M (1993) Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes & development 7:2135-2148.

Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M and Hotamisligil GS (2002) A central role for JNK in obesity and insulin resistance. Nature 420:333-336.

Howard A and Pelc SR (1953) Synthesis of deoxyribonucleic acid in normal and irradiated cells and its relation to chromosome breakage. Heredity 6:261-273.

Hu Y, Su L and Snow ET (1998) Arsenic toxicity is enzyme specific and its affects on ligation are not caused by the direct inhibition of DNA repair enzymes. Mutation research 408:203-218.

Huang C, Li J, Ding M, Wang L, Shi X, Castranova V, Vallyathan V, Ju G and Costa M (2001) Arsenic-induced NFkappaB transactivation through Erks- and JNKs- dependent pathways in mouse epidermal JB6 cells. Molecular and cellular biochemistry 222:29-34.

Huang SC and Lee TC (1998) Arsenite inhibits mitotic division and perturbs spindle dynamics in HeLa S3 cells. Carcinogenesis 19:889-896.

Hyun JS, Satsu H and Shimizu M (2007) Cadmium induces interleukin-8 production via NF-kappaB activation in the human intestinal epithelial cell, Caco-2. Cytokine 37:26-34.

Ip YT and Davis RJ (1998) Signal transduction by the c-Jun N-terminal kinase (JNK)- -from inflammation to development. Current opinion in cell biology 10:205-219.

Jeong EM, Moon CH, Kim CS, Lee SH, Baik EJ, Moon CK and Jung YS (2004) Cadmium stimulates the expression of ICAM-1 via NF-kappaB activation in

194

cerebrovascular endothelial cells. Biochemical and biophysical research communications 320:887-892.

Jia JS, Xu SR, Ma J, Jia CR, Yao YR and Wang Y (2003) [Effect of arsenic trioxide on the expression of cyclins gene in HL60 cells]. Zhonghua nei ke za zhi [Chinese journal of internal medicine] 42:113-116.

Jing Y, Dai J, Chalmers-Redman RM, Tatton WG and Waxman S (1999) Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Blood 94:2102-2111.

Kaltreider RC, Pesce CA, Ihnat MA, Lariviere JP and Hamilton JW (1999) Differential effects of arsenic(III) and chromium(VI) on nuclear transcription factor binding. Molecular carcinogenesis 25:219-229.

Kamata H, Honda S, Maeda S, Chang L, Hirata H and Karin M (2005) Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 120:649-661.

Karin M, Liu Z and Zandi E (1997) AP-1 function and regulation. Current opinion in cell biology 9:240-246.

Kitamura M and Hiramatsu N (2010) The oxidative stress: endoplasmic reticulum stress axis in cadmium toxicity. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 23:941-950.

Kitchin KT (2001) Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicology and applied pharmacology 172:249-261.

Klein CB, Frenkel K and Costa M (1991) The role of oxidative processes in metal carcinogenesis. Chemical research in toxicology 4:592-604.

Kligerman AD and Tennant AH (2007) Insights into the carcinogenic mode of action of arsenic. Toxicology and applied pharmacology 222:281-288.

195

Kozul-Horvath CD, Zandbergen F, Jackson BP, Enelow RI and Hamilton JW (2012) Effects of low-dose drinking water arsenic on mouse fetal and postnatal growth and development. PloS one 7:e38249.

Kyriakis JM and Avruch J (1990) pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine. The Journal of biological chemistry 265:17355-17363.

Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J and Woodgett JR (1994) The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156-160.

Lau AT, Li M, Xie R, He QY and Chiu JF (2004) Opposed arsenite-induced signaling pathways promote cell proliferation or apoptosis in cultured lung cells. Carcinogenesis 25:21-28.

Lee JM, Li J, Johnson DA, Stein TD, Kraft AD, Calkins MJ, Jakel RJ and Johnson JA (2005) Nrf2, a multi-organ protector? FASEB journal : official publication of the Federation of American Societies for Experimental Biology 19:1061-1066.

Lee TC and Ho IC (1995) Modulation of cellular antioxidant defense activities by sodium arsenite in human fibroblasts. Archives of toxicology 69:498-504.

Lee TC, Huang RY and Jan KY (1985a) Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells. Mutation research 148:83-89.

Lee TC, Oshimura M and Barrett JC (1985b) Comparison of arsenic-induced cell transformation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis 6:1421-1426.

Lei W, Yu R, Mandlekar S and Kong AN (1998) Induction of apoptosis and activation of interleukin 1beta-converting enzyme/Ced-3 protease (caspase-3) and c-Jun NH2- terminal kinase 1 by benzo(a)pyrene. Cancer research 58:2102-2106.

Li G, Lee LS, Li M, Tsao SW and Chiu JF (2011) Molecular changes during arsenic- induced cell transformation. Journal of cellular physiology 226:3225-3232.

Li N and Karin M (1999) Is NF-kappaB the sensor of oxidative stress? FASEB journal : official publication of the Federation of American Societies for Experimental Biology 13:1137-1143.

196

Liu J and Waalkes M (2005) Focal adhesion kinase as a potential target in arsenic toxicity. Toxicological sciences : an official journal of the Society of Toxicology 84:212-213.

Liu SX, Athar M, Lippai I, Waldren C and Hei TK (2001) Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity. Proceedings of the National Academy of Sciences of the United States of America 98:1643-1648.

Lynn S, Gurr JR, Lai HT and Jan KY (2000) NADH oxidase activation is involved in arsenite-induced oxidative DNA damage in human vascular smooth muscle cells. Circulation research 86:514-519.

Maiti S and Chatterjee AK (2000) Differential response of cellular antioxidant mechanism of liver and kidney to arsenic exposure and its relation to dietary protein deficiency. Environmental toxicology and pharmacology 8:227-235.

Manna SK, Zhang HJ, Yan T, Oberley LW and Aggarwal BB (1998) Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappaB and activated protein- 1. The Journal of biological chemistry 273:13245-13254.

Menghini R (1988) Genotoxicity of active oxygen species in mammalian cells. Mutation research 195:215-230.

Mitchison JM (1971) The Biology of the Cell Cycle, Cambridge, UK, Cambridge University Press.

Navas-Acien A, Sharrett AR, Silbergeld EK, Schwartz BS, Nachman KE, Burke TA and Guallar E (2005) Arsenic exposure and cardiovascular disease: a systematic review of the epidemiologic evidence. American journal of epidemiology 162:1037- 1049.

Nguyen N, Bonzo JA, Chen S, Chouinard S, Kelner MJ, Hardiman G, Belanger A and Tukey RH (2008) Disruption of the ugt1 locus in mice resembles human Crigler- Najjar type I disease. The Journal of biological chemistry 283:7901-7911.

Nordenson I and Beckman L (1991) Is the genotoxic effect of arsenic mediated by oxygen free radicals? Human heredity 41:71-73.

197

Nurse P (2000) A long twentieth century of the cell cycle and beyond. Cell 100:71-78.

Oshimura M and Barrett JC (1986) Chemically induced aneuploidy in mammalian cells: mechanisms and biological significance in cancer. Environmental mutagenesis 8:129-159.

Oya-Ohta Y, Kaise T and Ochi T (1996) Induction of chromosomal aberrations in cultured human fibroblasts by inorganic and organic arsenic compounds and the different roles of glutathione in such induction. Mutation research 357:123-129.

Park WH, Seol JG, Kim ES, Hyun JM, Jung CW, Lee CC, Kim BK and Lee YY (2000) Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis. Cancer research 60:3065-3071.

Parsons JT (2003) Focal adhesion kinase: the first ten years. Journal of cell science 116:1409-1416.

Pi J, Diwan BA, Sun Y, Liu J, Qu W, He Y, Styblo M and Waalkes MP (2008) Arsenic-induced malignant transformation of human keratinocytes: involvement of Nrf2. Free radical biology & medicine 45:651-658.

Playford RJ, Marchbank T, Goodlad RA, Chinery RA, Poulsom R and Hanby AM (1996) Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage. Proceedings of the National Academy of Sciences of the United States of America 93:2137-2142.

Pontius FW, Brown KG and Chen CJ (1994) Health implication of arsenic in drinking water. J Am Water Works Assoc 86:52-63.

Pulverer BJ, Kyriakis JM, Avruch J, Nikolakaki E and Woodgett JR (1991) Phosphorylation of c-jun mediated by MAP kinases. Nature 353:670-674.

Qian Y, Castranova V and Shi X (2003) New perspectives in arsenic-induced cell signal transduction. Journal of inorganic biochemistry 96:271-278.

Qu W, Bortner CD, Sakurai T, Hobson MJ and Waalkes MP (2002) Acquisition of apoptotic resistance in arsenic-induced malignant transformation: role of the JNK signal transduction pathway. Carcinogenesis 23:151-159.

Ribieras S, Tomasetto C and Rio MC (1998) The pS2/TFF1 trefoil factor, from basic research to clinical applications. Biochimica et biophysica acta 1378:F61-77.

198

Rorke EA, Sizemore N, Mukhtar H, Couch LH and Howard PC (1998) Polycyclic aromatic hydrocarbons enhance terminal cell death of human ectocervical cells. International journal of oncology 13:557-563.

Roth W and Reed JC (2002) Apoptosis and cancer: when BAX is TRAILing away. Nature medicine 8:216-218.

Sabapathy K, Kallunki T, David JP, Graef I, Karin M and Wagner EF (2001) c-Jun NH2-terminal kinase (JNK)1 and JNK2 have similar and stage-dependent roles in regulating T cell apoptosis and proliferation. The Journal of experimental medicine 193:317-328.

Sakurai T, Maeda S, Chang L and Karin M (2006) Loss of hepatic NF-kappa B activity enhances chemical hepatocarcinogenesis through sustained c-Jun N-terminal kinase 1 activation. Proceedings of the National Academy of Sciences of the United States of America 103:10544-10551.

Salas VM and Burchiel SW (1998) Apoptosis in Daudi human B cells in response to benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol. Toxicology and applied pharmacology 151:367-376.

Samikkannu T, Chen CH, Yih LH, Wang AS, Lin SY, Chen TC and Jan KY (2003) Reactive oxygen species are involved in arsenic trioxide inhibition of pyruvate dehydrogenase activity. Chemical research in toxicology 16:409-414.

Schreck R, Albermann K and Baeuerle PA (1992a) Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review). Free radical research communications 17:221-237.

Schreck R, Meier B, Mannel DN, Droge W and Baeuerle PA (1992b) Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. The Journal of experimental medicine 175:1181-1194.

Schreck R, Rieber P and Baeuerle PA (1991) Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. The EMBO journal 10:2247-2258.

199

Sen CK and Packer L (1996) Antioxidant and redox regulation of gene transcription. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 10:709-720.

Sheikh MS, Hollander MC and Fornance AJ, Jr. (2000) Role of Gadd45 in apoptosis. Biochemical pharmacology 59:43-45.

Shibata W, Maeda S, Hikiba Y, Yanai A, Sakamoto K, Nakagawa H, Ogura K, Karin M and Omata M (2008) c-Jun NH2-terminal kinase 1 is a critical regulator for the development of gastric cancer in mice. Cancer research 68:5031-5039.

Shimada T, Inoue K, Suzuki Y, Kawai T, Azuma E, Nakajima T, Shindo M, Kurose K, Sugie A, Yamagishi Y, Fujii-Kuriyama Y and Hashimoto M (2002) Arylhydrocarbon receptor-dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered C57BL/6J mice. Carcinogenesis 23:1199-1207.

Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO, Herman JG, Baylin SB, Sidransky D, Gabrielson E, Brock MV and Biswal S (2006) Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS medicine 3:e420.

Soldani C and Scovassi AI (2002) Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis : an international journal on programmed cell death 7:321-328.

Strassburg CP, Kneip S, Topp J, Obermayer-Straub P, Barut A, Tukey RH and Manns MP (2000) Polymorphic gene regulation and interindividual variation of UDP- glucuronosyltransferase activity in human small intestine. The Journal of biological chemistry 275:36164-36171.

Strassburg CP, Nguyen N, Manns MP and Tukey RH (1999) UDP- glucuronosyltransferase activity in human liver and colon. Gastroenterology 116:149- 160.

200

Sugatani J, Nishitani S, Yamakawa K, Yoshinari K, Sueyoshi T, Negishi M and Miwa M (2005a) Transcriptional regulation of human UGT1A1 gene expression: activated glucocorticoid receptor enhances constitutive androstane receptor/pregnane X receptor-mediated UDP-glucuronosyltransferase 1A1 regulation with glucocorticoid receptor-interacting protein 1. Molecular pharmacology 67:845-855.

Sugatani J, Sueyoshi T, Negishi M and Miwa M (2005b) Regulation of the human UGT1A1 gene by nuclear receptors constitutive active/androstane receptor, pregnane X receptor, and glucocorticoid receptor. Methods in enzymology 400:92-104.

Sugiyama M (1994) Role of cellular antioxidants in metal-induced damage. Cell biology and toxicology 10:1-22.

Swift H (1950) The constancy of desoxyribose nucleic acid in plant nuclei. Proceedings of the National Academy of Sciences of the United States of America 36:643-654.

Tian J, Huang H, Hoffman B, Liebermann DA, Ledda-Columbano GM, Columbano A and Locker J (2011) Gadd45beta is an inducible coactivator of transcription that facilitates rapid liver growth in mice. The Journal of clinical investigation 121:4491- 4502.

Ueda A, Kakizaki S, Negishi M and Sueyoshi T (2002) Residue threonine 350 confers steroid hormone responsiveness to the mouse nuclear orphan receptor CAR. Molecular pharmacology 61:1284-1288.

Vahidnia A, van der Voet GB and de Wolff FA (2007) Arsenic neurotoxicity--a review. Human & experimental toxicology 26:823-832.

Vogt BL and Rossman TG (2001) Effects of arsenite on p53, p21 and cyclin D expression in normal human fibroblasts -- a possible mechanism for arsenite's comutagenicity. Mutation research 478:159-168.

Wang TS and Huang H (1994) Active oxygen species are involved in the induction of micronuclei by arsenite in XRS-5 cells. Mutagenesis 9:253-257.

Wang TS, Kuo CF, Jan KY and Huang H (1996) Arsenite induces apoptosis in Chinese hamster ovary cells by generation of reactive oxygen species. Journal of cellular physiology 169:256-268.

Watson JD and Crick FH (1953) Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171:737-738.

201

Wu JP, Chang LW, Yao HT, Chang H, Tsai HT, Tsai MH, Yeh TK and Lin P (2009) Involvement of oxidative stress and activation of aryl hydrocarbon receptor in elevation of CYP1A1 expression and activity in lung cells and tissues by arsenic: an in vitro and in vivo study. Toxicological sciences : an official journal of the Society of Toxicology 107:385-393.

Xie W, Barwick JL, Downes M, Blumberg B, Simon CM, Nelson MC, Neuschwander-Tetri BA, Brunt EM, Guzelian PS and Evans RM (2000) Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature 406:435-439.

Yamamoto Y, Moore R, Flavell RA, Lu B and Negishi M (2010) Nuclear receptor CAR represses TNFalpha-induced cell death by interacting with the anti-apoptotic GADD45B. PloS one 5:e10121.

Yamanaka K, Hasegawa A, Sawamura R and Okada S (1989) Dimethylated arsenics induce DNA strand breaks in lung via the production of active oxygen in mice. Biochemical and biophysical research communications 165:43-50.

Yamanaka K, Hoshino M, Okamoto M, Sawamura R, Hasegawa A and Okada S (1990) Induction of DNA damage by dimethylarsine, a metabolite of inorganic arsenics, is for the major part likely due to its peroxyl radical. Biochemical and biophysical research communications 168:58-64.

Yih LH, Ho IC and Lee TC (1997) Sodium arsenite disturbs mitosis and induces chromosome loss in human fibroblasts. Cancer research 57:5051-5059.

Yih LH and Lee TC (1999) Effects of exposure protocols on induction of kinetochore- plus and -minus micronuclei by arsenite in diploid human fibroblasts. Mutation research 440:75-82.

Yih LH and Lee TC (2000) Arsenite induces p53 accumulation through an ATM- dependent pathway in human fibroblasts. Cancer research 60:6346-6352.

Yih LH, Peck K and Lee TC (2002) Changes in gene expression profiles of human fibroblasts in response to sodium arsenite treatment. Carcinogenesis 23:867-876.

202

Yoshii S, Tanaka M, Otsuki Y, Fujiyama T, Kataoka H, Arai H, Hanai H and Sugimura H (2001) Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene. Molecular and cellular biology 21:6796-6807.

Yueh MF and Tukey RH (2007) Nrf2-Keap1 signaling pathway regulates human UGT1A1 expression in vitro and in transgenic UGT1 mice. The Journal of biological chemistry 282:8749-8758.

CHAPTER 4

General Conclusions

202 203

Numerous factors, including age (particularly the neonatal period), diet, disease states, drug-drug interactions (induction and inhibition), hormonal effects, ethnicity, genetic polymorphism, and protein-protein interactions can alter human

UGT activity (Miners and Mackenzie, 1991; de Wildt et al., 1999). This dissertation work has contributed to further identifying and understanding how these factors influence the metabolism and disposition of glucuronidated drugs. I have shown the functional relevance of UGT dimerization in human hepatocytes, as well as the ability of oral arsenic exposure to modulate UGT1A1 gene expression during neonatal development. Determining how such factors affect UGT regulation, function, binding affinity, and substrate specificity will benefit the field of pharmacology and toxicology, in both drug design and risk assessment, respectively. In conclusion, my findings reflect the complicated nature of UGTs, thus emphasizing the importance of valid systems by which to study them and most importantly, recognizing the significant consequences of interindividual variability in pharmacotherapy.

Future Implications of this Work

UGT Interactions in Human Hepatocytes

Glucuronidation is one of the major metabolic pathways that serves as an essential clearance mechanism for a myriad of compounds, including drugs, dietary chemicals, environmental pollutants, and endogenous compounds. The significant number of substrates that are capable of undergoing glucuronidation has resulted in increased attention towards development of in vitro tools by which to help predict in vivo glucuronidation (Miners et al., 2004; (Foti and Fisher, 2012). A simplified

204 system that allows for the expression of a single gene product is inherently attractive for mechanistic studies of DMEs or to examine mechanisms of drug-drug interactions.

However, it is important to understand the inherent difficulties and potential discrepancies that may arise in the use of these systems as predictors of in vivo xenobiotic metabolism (Remmel and Burchell, 1993). Tissues fractions (HLMs or

S9), fresh or cryopreserved hepatocytes, and recombinant UGT enzymes are the current systems utilized for studying glucuronidation in vitro. HLMs are generally considered the easiest to use since contributions from Phase I and Phase II enzymes can easily be determined by the selective addition of the necessary cofactors for each pathway (Fisher et al., 2002). Cryopreserved hepatocytes have been determined to provide the most accurate prediction of in vivo glucuronidation parameters from in vitro data, making them the most physiologically relevant system for studying in vitro activity (Soars et al., 2002; Engtrakul et al., 2005). Recombinant systems overexpressing one or multiple UGTs have also been utilized to study glucuronidation

(Coffman et al., 1995; Fujiwara et al., 2007; Nakajima et al., 2007). Unfortunately,

UGT expression in these artificial environments may differ from native cells and the enzymatic contribution of each UGT isoform is very difficult to isolate due to the fact that interactions vary depending on isoform, substrate, and expression ratio.

Additionally, post-translational modifications to the UGTs that have been shown to impact activity may not occur in these simplified expression systems (Miners et al.,

2006; Ishii et al., 2010). Differences in lipid components between preparation and source of synthesis, as well as general membrane circumstances have been implicated in affecting UGT interactions and may account for in vitro intrinsic clearances that

205 severely under-predict in vivo hepatic clearance (Soars et al., 2002; Miners et al.,

2004; Fujiwara et al., 2010). Insect-expressed systems, such as Supersomes, are another common method by which to study single UGT isoforms, but also lack the potential to exhibit the heterodimeric protein interactions exhibited in more complex systems. With accumulating evidence exemplifying discrepancies between recombinant systems and whole cell systems, it is necessary to address the use of siRNA knockdown as an alternative process for evaluating UGT enzymology. This work has provided confirmation of interactions previously noted for UGT1A9 and

UGT2B7 in over-expressed cellular systems and suggests that UGT-UGT interactions are physiologically relevant phenomena whose effects can be observed in human hepatocytes. While further characterization with additional UGT isoforms as well as other cellular components (CYPs, transporters) is still necessary, the current data supports that caution should be taken in utilizing some of the more simplified, in vitro

UGT systems in which heterodimeric protein interactions are unable to occur.

The clinical relevance of UGT interactions is not well studied, however, there is significant potential for impacting drug-induced toxicity. Previous observations have shown that heterodimerization of UGTs with mutant forms can lead to altered glucuronidation activity (Meech and Mackenzie, 1997; Koiwai et al., 1996; Levesque et al., 2007; Ito et al., 2002; Nagar et al., 2004). It is known that CN-I and CN-II are both autosomal recessively inherited conditions (Crigler and Najjar, 1952; Kadakol et al., 2000). This is why heterozygous carriers are still capable of maintaining normal bilirubin levels. However, Koiwai et al. observed that heterozygous carriers of certain

UGT1A1 mutations displayed mild to moderate levels of hyperbilirubinemia. This

206 suggests an autosomal dominant pattern of inheritance in which UGT1A1 mutants act as a dominant negative protein toward the functional UGT1A1 wild-type protein

(Koiwai et al., 1996). Dr. Operaña’s dissertation work also tested this theory. To determine the effects of homo/heterodimerization on UGT function, she co-expressed several N-terminal truncated forms of UGT1A1 with the full-length form. The results all showed significant decreases in wild-type function, suggesting that the inactive, truncated forms function as dominant negative proteins. It can therefore follow that an individual expressing a mutant form of UGT1A1 may be at greater risk of ADRs due to lowered activity of the dimeric UGTs (Operaña, 2008). Investigation of the functional consequences of UGT dimerization has broadened the complexity of pharmacogenetics. These findings complement my dimerization work in human hepatocytes and further indicate dimerization among UGT proteins as an important factor in the pharmacokinetics and pharmacodynamics of a drug.

Intestinal Microflora and UGT1A1 Induction

Following ingestion, inorganic arsenic (iAs) is predominantly excreted as the methylated metabolite, dimethylarsinic acid (DMAV) and to a lesser extent, monomethylarsonic acid (MMAV). The identification of these pentavalent methylated metabolites in animal urine after exposure implicates biomethylation as a major detoxification mechanism. However, the formation of reactive trivalent intermediates, monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) has forced researchers to reconsider methylation as an activation process. In addition, the discovery of new sulfur containing methylated As metabolites,

207 monomethylmonothioarsonic acid (MMMTAV) and dimethylmonothioarsinic acid

(DMMTAV) (Naranmandura et al., 2007; Raml et al., 2007) in human urine has provoked re-evaluation of arsenic biostransformation altogether (Hughes and Kenyon,

1998; Styblo et al., 2000; Rehman and Naranmandura, 2012). These findings place particular emphasis on the importance of speciation analysis in evaluating risk from arsenic exposure (Van de Wiele et al., 2010).

Although iAs may be the predominant form present in contaminated water and soils, the speciation changes that it undergoes during GI transit are not well characterized. The highly reducing environment as well as complex microbial community of the gut can greatly contribute to the pre-systemic (systemic being all metabolism carried out by human cells) biotransformation of ingested iAs (Kubachka et al., 2009). The importance of pre-systemic metabolism by the microbe-rich environment of the GI tract has been demonstrated with in vivo animal models and in vitro experiments with animal microbiota (Hall et al., 1997; Kubachka et al., 2009).

However, Van de Wiele et al. was the first to confirm these findings in humans, by exposing cultured bacteria from human intestine to iAs or four types of soils with arsenic naturally present. In the bacteria, MMAV and the highly toxic MMAIII were formed from both pure iAs and iAs present in soils. These results validate that bacteria living in the human gut can increase the toxicity of arsenic ingested from contaminated food and water (Van de Wiele et al., 2010).

Gut microbial communities represent one source of metabolic diversity, as shaping of the human microbial landscape is driven by a series of complex and dynamic interactions throughout life, including diet, life-style, disease, and antibiotic

208 use. This developmental trajectory of the microbiome plays a key role in shaping the metabolic phenotype of the host and greatly influences host biochemistry and susceptibility to disease (Nicholson et al., 2012; Yatsunenko et al., 2012). Therefore, the unique environment of the gut, more specifically pre-systemic metabolism, could be a contributing factor in influencing UGT1A1 induction patterns observed with oral arsenic exposure. Antibiotic treatment of hUGT1 mice could aid in addressing the influence of intestinal microflora on activity and expression of DMEs in response to arsenic exposure.

References

Crigler JF, Jr. and Najjar VA (1952) Congenital familial nonhemolytic jaundice with kernicterus. Pediatrics 10:169-180. de Wildt SN, Kearns GL, Leeder JS and van den Anker JN (1999) Glucuronidation in humans. Pharmacogenetic and developmental aspects. Clinical pharmacokinetics 36:439-452.

209

Fujiwara R, Nakajima M, Oda S, Yamanaka H, Ikushiro S, Sakaki T and Yokoi T (2010) Interactions between human UDP-glucuronosyltransferase (UGT) 2B7 and UGT1A enzymes. Journal of pharmaceutical sciences 99:442-454.

Fujiwara R, Nakajima M, Yamanaka H, Katoh M and Yokoi T (2007) Interactions between human UGT1A1, UGT1A4, and UGT1A6 affect their enzymatic activities. Drug metabolism and disposition: the biological fate of chemicals 35:1781-1787.

Hall LL, George SE, Kohan MJ, Styblo M and Thomas DJ (1997) In vitro methylation of inorganic arsenic in mouse intestinal cecum. Toxicology and applied pharmacology 147:101-109.

Hughes MF and Kenyon EM (1998) Dose-dependent effects on the disposition of monomethylarsonic acid and dimethylarsinic acid in the mouse after intravenous administration. Journal of toxicology and environmental health Part A 53:95-112.

Ishii Y, Nurrochmad A and Yamada H (2010) Modulation of UDP- glucuronosyltransferase activity by endogenous compounds. Drug metabolism and pharmacokinetics 25:134-148.

Ito M, Yamamoto K, Maruo Y, Sato H, Fujiyama Y and Bamba T (2002) Effect of a conserved mutation in uridine diphosphate glucuronosyltransferase 1A1 and 1A6 on glucuronidation of a metabolite of flutamide. European journal of clinical pharmacology 58:11-14.

Koiwai O, Aono S, Adachi Y, Kamisako T, Yasui Y, Nishizawa M and Sato H (1996) Crigler-Najjar syndrome type II is inherited both as a dominant and as a recessive trait. Human molecular genetics 5:645-647.

Kubachka KM, Kohan MC, Herbin-Davis K, Creed JT and Thomas DJ (2009) Exploring the in vitro formation of trimethylarsine sulfide from dimethylthioarsinic acid in anaerobic microflora of mouse cecum using HPLC-ICP-MS and HPLC-ESI- MS. Toxicology and applied pharmacology 239:137-143.

Levesque E, Delage R, Benoit-Biancamano MO, Caron P, Bernard O, Couture F and Guillemette C (2007) The impact of UGT1A8, UGT1A9, and UGT2B7 genetic

210 polymorphisms on the pharmacokinetic profile of mycophenolic acid after a single oral dose in healthy volunteers. Clinical pharmacology and therapeutics 81:392-400.

Meech R and Mackenzie PI (1997) UDP-glucuronosyltransferase, the role of the amino terminus in dimerization. The Journal of biological chemistry 272:26913- 26917.

Miners JO and Mackenzie PI (1991) Drug glucuronidation in humans. Pharmacology & therapeutics 51:347-369.

Nagar S, Zalatoris JJ and Blanchard RL (2004) Human UGT1A6 pharmacogenetics: identification of a novel SNP, characterization of allele frequencies and functional analysis of recombinant allozymes in human liver tissue and in cultured cells. Pharmacogenetics 14:487-499.

Naranmandura H, Suzuki N, Iwata K, Hirano S and Suzuki KT (2007) Arsenic metabolism and thioarsenicals in hamsters and rats. Chemical research in toxicology 20:616-624.

Nicholson JK, Holmes E, Kinross J, Burcelin R, Gibson G, Jia W and Pettersson S (2012) Host-gut microbiota metabolic interactions. Science 336:1262-1267.

Operaña TN (2008) A tale of two drug metabolizing enzymes : the CYP1A1-GFP transgenic mouse and the promiscuous nature of UGTs, in Biomedical Sciences, UC San Diego.

Raml R, Rumpler A, Goessler W, Vahter M, Li L, Ochi T and Francesconi KA (2007) Thio-dimethylarsinate is a common metabolite in urine samples from arsenic-exposed women in Bangladesh. Toxicology and applied pharmacology 222:374-380.

Rehman K and Naranmandura H (2012) Arsenic metabolism and thioarsenicals. Metallomics : integrated biometal science.

211

Soars MG, Burchell B and Riley RJ (2002) In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance. The Journal of pharmacology and experimental therapeutics 301:382-390.

Styblo M, Del Razo LM, Vega L, Germolec DR, LeCluyse EL, Hamilton GA, Reed W, Wang C, Cullen WR and Thomas DJ (2000) Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Archives of toxicology 74:289-299.

Van de Wiele T, Gallawa CM, Kubachka KM, Creed JT, Basta N, Dayton EA, Whitacre S, Du Laing G and Bradham K (2010) Arsenic metabolism by human gut microbiota upon in vitro digestion of contaminated soils. Environmental health perspectives 118:1004-1009.

Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, Magris M, Hidalgo G, Baldassano RN, Anokhin AP, Heath AC, Warner B, Reeder J, Kuczynski J, Caporaso JG, Lozupone CA, Lauber C, Clemente JC, Knights D, Knight R and Gordon JI (2012) Human gut microbiome viewed across age and geography. Nature 486:222-227.